Genetic features of circular bacteriocins produced by Gram-positive bacteria

REVIEW ARTICLE

Genetic features of circular bacteriocins produced by Gram-positive bacteria ´ ´ ´ ´ Mercedes Maqueda, Marina Sanchez-Hidalgo, Matilde Fernandez, Manuel Montalban-L opez, Eva Valdivia & Manuel Mart´ınez-Bueno Departmento de Microbiolog´ıa, Facultad de Ciencias, Universidad de Granada, Granada, Spain

Correspondence: Mercedes Maqueda, Departamento de Microbiolog´ıa, Facultad de Ciencias, Universidad de Granada, Fuentenueva s/n, E-18071 Granada, Spain. Tel.:/fax: 134 958 242857; e-mail: [email protected] Received 15 June 2007; revised 26 July 2007; accepted 13 August 2007. First published online 22 November 2007. DOI:10.1111/j.1574-6976.2007.00087.x Editor: Ramon Diaz Orejas Keywords bacteriocin; processing; circularization; immunity determinants.

Abstract This review highlights the main genetic features of circular bacteriocins, which require the co-ordinated expression of several genetic determinants. In general terms, it has been demonstrated that the expression of such structural genes must be combined with the activity of proteins involved in maturation (cleavage/ circularization) and secretion outside the cell via different transporter systems, as well as multifaceted immunity mechanisms essential to ensuring the bacteria’s selfprotection against such strong inhibitors. Several circular antibacterial peptides produced by Gram-positive bacteria have been described to date, including enterocin AS-48, from Enterococcus faecalis S-48 (the first one characterized), gassericin A, from Lactobacillus gasseri LA39, and a similar one, reutericin 6, from Lactobacillus reuteri LA6, butyrivibriocin AR10, from the ruminal anaerobe Butyrivibrio fibrisolvens AR10, uberolysin, from Streptococcus uberis, circularin A, from Clostridium beijerinckii ATCC 25752, and subtilosin A, from Bacillus subtilis. We summarize here the progress made in the understanding of their principal genetic features over the last few years, during which the functional roles of circular proteins with wide biological activity have become clearer.

Introduction The phenomenon of microbial antagonism mediated by peptides has gained considerable attention in recent years because of their rapid bactericidal activity over a broad spectrum and the low propensity of bacteria to develop resistance against them. Antimicrobial peptides differ considerably in their primary structure although they are all normally fairly short molecules (12–100 amino acids) and are very often amphiphilic (Hancock & Diamond, 2000). They tend to kill their target cells by permeabilizing the cell membrane (Nissen-Meyer & Nes, 1997), although novel modes of action such as the inhibition of nucleic acid synthesis, protein synthesis, enzyme activity and cell-wall synthesis have recently been described (Brogden, 2005). Although their primary function was believed to be antimicrobial, it is now becoming clear that these peptides have a wide repertoire of activities that do not function solely against microorganisms and have interesting repercussions on the immune system (Jenssen et al., 2006). Interestingly, this antimicrobial arsenal has been isolated from almost all 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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species of life – prokaryotic and eukaryotic microorganisms, plants, insects and other invertebrates, fish, amphibians, birds and mammals, including humans – and it has been proposed that they represent crucial components of their immediate nonspecific defence against infections, protecting the host from virtually all kinds of invading bacteria, enveloped viruses and fungi (reviewed by Jenssen et al., 2006), which all have a high electric potential in their cytoplasmic membrane. Bacteriocins stand out among the wide variety of antimicrobial ribosomal peptides synthesized by bacteria. They have been found in all major lineages of Bacteria (revised by Riley & Wertz, 2002), and some members of the Archaea have also been seen to produce similar antimicrobial proteins (O’Connor & Schand, 2002). According to Klaenhammer (1988), 99% of all bacteria may produce at least one bacteriocin and the only reason why researchers have not isolated more is that one has not been looking for them. Bacteriocidal proteins produced by Gram-negative bacteria, e.g. the colicins produced by Escherichia coli, are often larger than 20 kDa and inhibit the growth of closely related strains FEMS Microbiol Rev 32 (2008) 2–22

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Genetics of circular bacteriocins

(Konisky, 1982). Colicin V and the Microcins, a heterogeneous group of small peptides (410 kDa) produced by enterobacterial strains, are exceptions. Microcins can be distinguished from colicins by their smaller size and the fact that their synthesis, nonlethal for the producer strains, is not SOS inducible (Moreno et al., 1995). In contrast to Gramnegative bacteria, Gram-positive bacteria usually produce bacteriocins smaller than 8 kDa, which, because of their amphipathic properties, are often membrane-permeabilizing peptides. In this way, they resemble many of the antimicrobial peptides produced by eukaryotes, although they are less self-toxic. These bacteriocins have so far been grouped into two major classes, which display great diversity with regard to their mode of action, structure, genetics, mode of secretion and choice of target organisms (Nes et al., 2007): class I: lantibiotics; which are small, posttranslationally modified peptides that contain unusual amino acids such as lanthionine, and class II: the heat-stable nonlantibiotics. A third class of bacteriocins has been suggested that includes secreted heat-labile, cell-wall degrading enzymes, but the classification of such enzymes as being bacteriocins has recently been thrown into doubt (Cotter et al., 2005a). In contrast to this, Heng et al. (2007) propose to retain this class for large bacteriocins, which is subdivided into IIIa (bacteriolysins) and IIIb (nonlytic proteins). The lantibiotics have been divided into two subgroups: type A, elongated molecules with a flexible structure in solution, and type B, which tend to have a more rigid, globular structure (although there are some exceptions). Lack of consensus also exists in the distinction between subgroups of the nonlantibiotics belonging to class II, which include a large number of small peptides that only have in common their marked thermostability and a lack of modified residues (Cleveland et al., 2001; Eijsink et al. 2002; Ross et al., 2002; Cotter et al., 2005a; Drider et al., 2006). In this new classification scheme, Nes et al. (2007) subdivided class II into four subclasses: IIa (antilisteria pediocin-like bacteriocins), IIb (two-peptide bacteriocins), IIc (leaderless peptide-

bacteriocins) and class IId (circular bacteriocins). Nevertheless, according to the recommendations of several research groups (Kemperman et al., 2003b; Kawai et al., 2004b; Maqueda et al., 2004; Heng & Tagg, 2006; Heng et al., 2007), the circular, posttranslationally modified bacteriocins deserve to be upgraded to a new class IV.

The circular bacteriocins: description of the gene clusters Over the past decade, different research groups have described this unique family of active proteins, isolated from very diverse sources, in which the N- and C-ends are linked to form a circular backbone. These studies constitute an ongoing prolific area of research (Trabi & Craik, 2002; Craik et al., 2003, 2006; Cole et al., 2004; Kalkum et al., 2004; Maqueda et al., 2004; Selsted et al., 2004; Hancock & Sahl, 2006). In accordance with Craik (2004), the term ‘circular’ has been adopted to distinguish this special group of geneencoded proteins from the classic ‘cyclic’ peptides also produced by microorganisms via multiple steps of enzymatic synthesis. Of particular interest among the most typical circular bacteriocins described (Table 1) are the peptide enterocin AS-48 (reviewed by Maqueda et al., 2004), which is homologous with no other bacteriocin described so far, except circularin A, produced by Clostridium beijerinckii ATCC 25752 (Kemperman et al., 2003b; Kawai et al., 2004b) gassericin A and reutericin 6, two hydrophobic bacteriocins produced by species from Lactobacillus, which show 98% similarity to acidocin B, produced by Lactobacillus acidophilus (Kawai et al., 2004a). Others include butyrivibriocin AR10, produced by the anaerobic ruminal bacterium Butyrivibrium fibrisolvens AR10 (Kalmokoff et al., 2003), which displays significant amino acid similarity to gassericin A (Leer et al., 1995; Kalmokoff et al., 2003), uberolysin, the first circular bacteriocin to be characterized (biochemically and genetically) from Streptococcus uberis (Wirawan et al.,

Table 1. Characteristics of circular bacteriocins from Gram-positive bacteria Bacteriocin Class IV AS-48 Gassericin A Reutericin 6 Acidocin B Butyrivibriocin AR10 Uberolysin Circularin A Class I Subtilosin A

Source

Leader

Amino acid (mass in Da)

Reference

Enterococcus faecalis S-48 Lactobacillus gasseri LA39 Lactobacillus reuteri LA6 Lactobacillus acidophilus M46 Butyrivibrium fibrisolvens AR10 Streptococcus uberis 42 Clostridium beijerinckii ATCC 25752

35 33 33 33 22 3 6

70 (7150) 58 (5652) 58 (5652) 58 (5620) 58 (5981.5) 78 (7048) 69 (ND)

´ Galvez et al. (1986) Kawai et al. (1998) Toba et al. (1991a) Leer et al. (1995) Kalmokoff & Teather (1997) Wirawan et al. (2007) Kemperman et al. (2003a)

8

35 (3399.7)

Kawulka et al. (2003)

Bacillus subtilis

Proposed Mw.

ND, not determined.

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2007), and the only circular lantibiotic described to date, and subtilosin A, produced by Bacillus subtilis (Marx et al., 2001; Kawulka et al., 2004). These bacteriocins range in size from 58 to 78 amino acids and are produced by Grampositive bacteria via gene translation. Their only difference from conventional linear proteins is that the gene-encoded precursor protein must be posttranslationally modified to join their termini to produce a seamless circle of peptide bonds. The structural details of subtilosin A (Fig. 1a) (Marx et al., 2001; Kawulka et al., 2003, 2004) and AS-48 (Fig. 1b), obtained by nuclear magnetic resonance (NMR) (Gonz´alez et al., 2000) and X-ray diffraction (S´anchez-Barrena et al., 2003), reveal a well-folded three-dimensional structure with a-helix motifs repeated regularly in their secondary structure, but no notable differences from those of their linear counterparts. Cross-linking the termini of a chain decreases the conformational entropy of the disordered and highly

flexible linear polypeptides, thus stabilizing their bioactive conformation and rendering them more resistant to proteolysis due to a lack of exopeptidase cleavage sites. Previous reviews in this field have focused on the biochemical and structural aspects of this new generation of proteins. For the sake of introduction and continuity this review refers to some of these earlier works but deals primarily with the more recent aspects of genetic research. A comparative analysis of their genetic determinants leads to the recognition of complex but common features involved in the expression and secretion of these inhibitors and also immunity against them. Their complexity, together with the biosynthetic resources necessary to produce such circular proteins, must have evolved because of the advantages conferred on them by circularization, i.e. increased resistance to protease digestion and enhanced thermodynamic stability and integrity in the protein structure, both of which

(a)

E

E D

K 4

D

D

W

1

7

K

13

G A A C L V D G G P W L G F L G T A G A I E F D P I N K G C A T C S I

34

(b)

31

D

28

1– 5

W

22

9 – 21

25–34

37–45

51–62

64–70

MAKEFGIPAAVAGTVLNVVEAGGWVTTIVSILTAVGSGGLSLLAAAGRESIKAYLKKEIKKKGKRAVIAW

DF-I

DF-II

´ Fig. 1. Three-dimensional structure of (a) subtilosin A (adapted from Marx et al., 2001) and (b) AS-48 (Gonzalez et al., 2000) and (resolved by NMR). Proposed models depicting the topology of subtilosin A embedded in the hydrophilic/hydrophobic interface of the lipid bilayer, using subtilosin ´ A-fluorescence and solid-state NMR data (adapted from Thennarasu et al., 2005) and AS-48 as shown by X-ray diffraction (adapted from SanchezBarrena et al., 2003).

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Genetics of circular bacteriocins

seem to improve their biological activity in vivo. In fact, due to their wide spectrum of action and stability across considerable pH and temperature ranges, they constitute one of the most attractive groups of inhibitors described to date. We update the composition of the group and define the common properties of these circular bacteriocins before highlighting the most recent observations regarding the transcriptional regulation of their genes, together with the biosynthetic machinery and characterization of immunity proteins.

Enterocin AS-48 Biochemical and biological characteristics The broad-spectrum AS-48 is a cationic, 70-residue, circular peptide produced during the exponential growth of Enterococcus faecalis ssp. liquefaciens strain S-48 (Ga´ lvez et al., 1986). Several natural AS-48 variants – enterocin EFS2, enterocin 4 and bacteriocin 21 – have subsequently been reported for other strains of Enterococcus faecalis (Joosten et al., 1996; Maisnier-Patin et al., 1996; Tomita et al., 1997) as well as for Enterococcus faecium 7C5 (Folli et al., 2003). More recently, an AS-48 variant, AS-48RJ, identical to AS48, except for Glu20, which is replaced by Val, has been characterized from an Enterococcus faecium strain isolated from home-made goat cheese (Abriouel et al., 2005). Enterocin AS-48 exhibits bactericidal activity against a wide variety of Gram-positive bacteria, including food-spoilage and pathogenic Gram-positive bacteria such as Bacillus, Clostridium, Brochothrix thermosphacta, Staphylococcus aureus and Listeria monocytogenes (Ga´ lvez et al., 1989a; Mendoza et al., 1999; Abriouel et al., 2002) and also against some Gram-negative species (Ga´ lvez et al., 1989b; Abriouel et al., 1998). The inhibitory activity of the enterocin is enhanced when applied in combination with other physical or chemical treatments such as heat, organic acids, nitrites or chelating agents (Abriouel et al., 2002; Ananou et al., 2004). These treatments also extend their inhibitory spectrum to intrinsically resistant Gram-negative bacteria such as Salmonella choleraesuis and Escherichia coli O157 (Abriouel et al., 1998; Ananou et al., 2005). The numerous studies carried out on this bacteriocin have helped to clarify its molecular composition and structure (reviewed by Maqueda et al., 2004). Analysis of the composition of purified AS-48 reveals a high proportion of basic amino acids, which give it a strong basic character with a pI of nearly 10.5. It does not contain lanthionine, b-methyllanthionine or dehydrated residues, thus making it clearly different from lantibiotics and defensins in that it lacks disulphide bridges, and so its additional stability must derive from its circular backbone (Cobos et al., 2001). Determination of the DNA sequence of the AS-48 structural FEMS Microbiol Rev 32 (2008) 2–22

gene (Mart´ınez-Bueno et al., 1994), the sequences of its protease digestion fragments and complementary massspectrometry results have led to the unambiguous identification of its circular structure. This was the first example of a posttranslational modification described in which a circular structure arises from a ‘head-to-tail’ linkage (Samyn et al., 1994). Reports of the complete 1H NMR assignment of AS-48 proton resonances, together with the resulting secondary structural pattern, were essential for the determination of the high-resolution, three-dimensional solution structure resolved by NMR (Gonz´alez et al., 2000) (Fig. 1b). AS-48 folds in an all-a-helix manner with five helical regions spanning residues 9–21 (a1), 25–34 (a2), 37–45 (a3), 51–62 (a4) and 64–5 (a5) (Langdon et al., 1998; Gonza´ lez et al., 2000) (Fig. 1b). The backbone circularization of the AS-48 precursor occurs in the middle of helix a5 and appears to confer considerable stability upon the three-dimensional structure, the thermal denaturation temperature of which is 102 1C (Cobos et al., 2001). Enterocin AS-48 interacts with the cytoplasmic membrane of susceptible bacteria, into which it inserts itself in a voltage-independent manner, inducing ion permeation accompanied by a collapse of membrane potential. As has been suggested, pore formation may require a certain distribution of the electrostatic and/or hydrophobic surfaces, and an analysis of structure–function relationships is essential to a final understanding of the interaction between AS-48 and cell membranes. The binding of negatively charged membranes may be initiated by the high positive electrostatic potential of the polypeptides (cf. review Maqueda et al., 2004). Thus, the positive charges in the side chains of the residues in helix a4 and in the turn linking helix a4 to helix a5 form a cluster that might determine its antibacterial activity by promoting the formation of pores in cell membranes. Analysis of the oligomeric structure found in AS-48 crystals, however, probes deeper into the mechanism of its antibacterial activity, which involves a transition from the water-soluble DF-I to the membrane-bound DF-II at the membrane surface (Fig. 1b), implying an effective insertion into the membrane that involves a structural reorganization of a hydrosoluble dimeric form of AS-48 (Sa´ nchez-Barrena et al., 2003). In fact, the results obtained with a 21-residue peptide fragment of bacteriocin AS-48 containing only its putative membrane-interacting region confirm that the mechanism of membrane disruption by this bacteriocin is more complicated than one merely driven by a deposition of positively charged molecules on the bacterial membrane. Some other regions of the protein must be present, such as, for instance, the hydrophobic patches surrounding the dimer DF-II, to enhance the accumulation of the peptide and favour membrane permeation (Jim´enez et al., 2005). 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Sequence analysis of the as-48 region The as-48 gene cluster for enterocin AS-48 and the identical bacteriocin 21 are located on conjugative, pheromoneresponse plasmids pMB2 (68 kb) (Mart´ınez-Bueno et al., 1990) and pPD1 (59 kb) (Tomita et al., 1997), respectively, both from Enterococcus faecalis strains. The only exception is the AS-48RJ variant, from Enterococcus faecium, in which the as-48 gene cluster is located on the bacterial chromosome (Abriouel et al., 2005). Interestingly, analogous genetic information (pBt136–140 determinants) involved in the production and export of a peptide similar in length and sequence to the processed propeptide of AS-48 has been found in pBtoxis, a toxin-coding plasmid of Bacillus thuringiensis ssp. israelensis (Berry et al., 2002), although no study has been undertaken into the possible production of circular peptide antibiotics at the protein level. The as-48A structural gene encodes a 105-amino-acid prepeptide consisting of a 35-amino-acid signal peptide and a 70-aminoacid propeptide, coinciding with the previously determined AS-48 amino-acid sequence described by Mart´ınez-Bueno et al. (1994) (Fig. 2). The signal peptide contains a typical (a)

positively charged N terminus (four residues) followed by a stretch of hydrophobic residues and a polar region next to the cleavage site. The data presented in Fig. 2 show that AS-48 maturation requires a series of modifications during secretion via the following events: the AS-48-translated primary product is a proprepeptide of 105-amino-acid residues processed between H 1 and M11 by a specific peptidase to produce a 70-amino-acid prepeptide, which must undergo a new posttranslational change that results in the linking of the N-terminal methionine (M11) to the C-terminal tryptophan (W170), with the elimination of a water molecule, to result in the active and mature AS-48 molecule (Mart´ınez-Bueno et al., 1994) (Fig. 2). According to the results published by Mart´ınez-Bueno et al. (1998) and D´ıaz et al. (2003), the full expression of AS48 and immunity to AS-48 depend on the co-ordinated expression of ten genes: as-48A, B, C, C1, D, D1, E, F, G and H (Fig. 3) (GenBank accession number X79542). The characteristics of the DNA sequence analysis and genes identified are described in Table 2. Interestingly, the two as-48BC overlapping genes are located 73 nt downstream of the TAA termination codon of the as-48A structural gene, Prepeptide (n-residues)

Leader peptide -1 +1

Gassericin A

:

: Acidocin B AS-48 Subtilosin A Butyrivibriocin AR10 Circularin A Uberolysin

(b) a) Proteolytic cleavage between H

and M

Pro-peptide (70 residues) MAKEFGIPAAVAGTVLNVVEAGGWVTTIVSILTAVGSGGLSLLAAAGRESIKAYLKKEIKKKGKRAVIAW

b) Peptide bond formation between M

and W

AS-48 mature protein

Fig. 2. (a) Amino-acid sequence of the different prepeptides deduced from the nucleotide sequence of the respective structural genes. (b) Hypothetical maturation procedure for prepeptides is represented by AS-48 maturation. First, there is a proteolytic cleavage reaction between H 1 and M11, followed by peptide bond formation between M11 and W170 to yield the mature, circular AS-48 peptide head-to-tail link.

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Genetics of circular bacteriocins

B

A

as-48

C1

C

E

D

IR

F

G

H

D1

acd

1 B

3 4 IR

bvi

ubl

1

2

B

ComE

PurH

C

B

A

A

D

C

D

E

DR

cfgR

cir

DR

cfgK

A cfg01 cfg02

A

sbo/alb ywhB

6

E

D

C

B

E E

G

H

I

IR

C

DD

E

F

G

ywhl

IR

sboX sboA

albB

Fig. 3. Organization of the gene cluster involved in the production of and immunity to the circular bacteriocins AS-48 (Mart´ınez-Bueno et al., 1998; D´ıaz et al., 2003), acidocin B (Kawai et al., 2004), butyrivibriocin AR10 (Kalmokoff et al., 2003), uberolysin (Wirawan et al., 2007), circularin A (Kemperman et al., 2003b) and subtilosin A (Zheng et al., 2000). Structural genes encoding prebacteriocins (as-48A, acdB, bviA, ublA, cirA, sboA) are shown in red, putative biosyntethic and procesing genes (as-48B, ublB, cirB, albA, albE, albF) in blue and immunity genes (as-48D1, bviE, cirE, ublE, albB) in yellow. The ABC transporters involved in secretory functions (as-48C1D, ublCD, cirBD, albCD) are in green, while those involved in immunity (as48EFGH and the proposed cirGHI) are striped in yellow. Known regulatory genes are in brown while white genes have nonrelated functions. Putative promoters are shown by vertical arrows. Red arrows under a gene cluster indicate the synthetic mRNAs described, and the relative transcript abundance is indicated by the thickness of the arrow below (thicker lines corresponding to the more abundant mRNAs detected in Northern blottings).

separated by a characteristic inverted repeat (IR) with an estimated DG energy of 22.4 kcal mol 1 and the capacity to form a complex secondary structure (Fig. 4), which was initially thought to serve as a transcription terminator signal (Mart´ınez-Bueno et al., 1998). Nevertheless, recent results from site-directed mutagenesis experiments, in which three bases within the IR were replaced, have demonstrated that this structure represents a characteristic substrate for endoribonucleases involved in mRNA processing and is essential for the controlled expression of the as-48BC genes (Fern´andez et al., 2008). Moreover, the following modules of genes FEMS Microbiol Rev 32 (2008) 2–22

described in the as-48 cluster also overlap: as-48C1DD1, followed by an intergenic region of 204 nt, after which come the contiguous as-48EFGH genes, followed by a strong transcriptional terminator sequence (Mart´ınez-Bueno et al., 1998; D´ıaz et al., 2003). Functional and transcriptional DNA analysis of the as-48 cluster Most of the gene products involved in AS-48 production and immunity are predominantly basic, hydrophobic 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Table 2. Characteristics of predicted proteins encoded by the as-48 gene cluster (adapted from Mart´ınez-Bueno et al., 1998; D´ıaz et al., 2003) Gene

Amino acid

pI

Relevant characteristics

Putative protein function

as-48A as-48B as-48C as-48C1 as-48D as-48D1 as-48E as-48F

105 563 178 164 219 56 169 407

10.5 10.48 10.07 10.96 9.3 10.81 10.2 5.4

as-48G as-48H

227 399

7.8

Prepropeptide Integral membrane protein (12 TM) Integral membrane protein (4 TM) Integral membrane protein (6 TM) ATP-binding protein 2 TM Integral membrane protein (4 TM) Hydrophilic except for the N-terminal segment. Coiled-coil domain. Hydrophilic ATP-dependent protein Integral membrane protein (4 TM)

AS-48-precursor Biosynthetic machinery? Auxiliar immunity protein ABC transporter export ABC transporter export Immunity protein ABC transporter immunity Auxiliary protein involved in As-48G recycling ABC transporter immunity ABC transporter immunity

TM, transmembrane domains.

as48A-B

ATG-308nt-TAATTTAACAATATGATAAAAAAACAGGATATTTTCTAGAGATATTCTGTTTTTTAATTA

acdB-ORF3 ATG-200nt-TAGTGATTATAAAGCTTTATGAATAGACTATTCAAAAAAGGCTAATAGGTATTAATACCTATTAGCCTTTTTCTT

cirA-B ATG-213nt-TAATCAAAATTTATGATTTTAATTAAATGGATAGCTTGCTACAGACAGGCTATCTATTTAATTAAATGAA

sboA-albA ATG-124nt-TAAGGATCTCCCAAAAGGGCATAGTCATTCTACTATGCCCTTTTTAAG

as-48* ∆G= − 22.4

acd

cir

sbo/alb†

∆G= − 25.1

∆G= − 23.6

∆G= − 23.4

Fig. 4. Sequence of the intergenic regions between the structural and the following genes in as-48, acd, cir and sbo/alb clusters. Stem-loop structures ´ predicted using the MFOLD program (Zuker & Mathews, 1999) (, adapted from Fernandez et al., 2008; w, adapted from Zheng et al., 1999). DG is the free 1 energy of secondary-structure formation (in kcal mol ).

proteins, several of which have a high number of potential transmembrane domains (TM) (twelve in As-48B, six in As48C1 and four in As-48C, As-48E and As-48H). Accurate predictive analytical methods have been used to investigate the possible function of As-48B, a large, hydrophobic protein (563 residues, pI of 10.48) previously described by Mart´ınez-Bueno et al. (1998). The existence of similar internal sequences detected in this protein seems to indicate a putative duplication in the molecule, which would imply a structure of 616 helical spanning segments. The homologies 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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found have been only with membrane proteins involved in transport or pore formation (unpublished results), in which the identification of domains and functional assignment of the sequences were not available. Nevertheless, phenotypic studies carried out upon the as-48ABC operon using mutagenesis with Tn5 revealed that inactivation of as-48B unambiguously prevented AS-48 production, thus confirming that it plays an essential part in AS-48 biogenesis. Disruption of the neighbouring as-48C gene, which encodes a hydrophobic 178-amino-acid protein (Table 2), decreases FEMS Microbiol Rev 32 (2008) 2–22

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Genetics of circular bacteriocins

AS-48 production and resistance (Ferna´ ndez et al., 2008). Using homology comparisons, Mart´ınez-Bueno et al. (1998) determined that the As-48D protein encodes the functional domain of an ATP-binding cassette (ABC) transporter, and As-48C1 (with six TMs) constitutes the membrane-spanning domain for this ABC transporter. On the basis of the phenotypes of different insertion mutants, D´ıaz et al. (2003) concluded that the as-48C1D transporter was devoted to exporting newly synthesized bacteriocin and providing low levels of immunity. The function of this transporter cannot be replaced by the second multicomponent ABC transporter, As-48EFGH, which is clearly involved in self-protection against exogenously administered AS-48 and works as a complementary immunity mechanism (D´ıaz et al., 2003). The expression of the as-48 gene cluster has been studied by Northern blotting using total RNA extracted from several JH2-2 transformants (Fig. 3). Two mRNA populations (T1 and T2 transcripts) were detected in the transcriptional analysis, thus ruling out the possibility that the as-48 genes are transcribed as a single operon. Nevertheless, transcript T1, driven by the promoter PA, transcribes the as-48ABC genes (TABC) and undergoes endonucleolytic cleavage, involving the accumulation of a very stable mRNA degradation product (TA) and a TBC with a very short lifespan, thus making its detection almost impossible (Fig. 3). The reasons why the as-48ABC genes are cotranscribed must be investigated through the functional analysis of their gene products in the cell: As-48B alone or in association with the As-48C1D translocation complex might form a pore to facilitate the exit of AS-48 molecules (Mart´ınez-Bueno et al. 1998), while the integral membrane protein As-48C is involved in protection functions (Ferna´ ndez et al., 2008). Apart from this, the long T2 transcript (c. 6.4 kb) transcribes the last seven genes (as-48C1DD1EFGH), although a second and shorter mRNA (T3, 5.4 kb) also translates the last four genes (as-48EFGH), a fact that confirms the existence of an internal promoter (D´ıaz et al., 2003). All in all, the results of D´ıaz et al. (2003) corroborate the importance of this second set of gene products in the cell, the constitutive expression of which is safeguarded by the two mRNAs T2 and T3. Finally, it is worth highlighting that a quorumsensing mechanism similar to those described in most of the bacteriocin systems characterized to date, lantibiotics (reviewed by Kleerebezem, 2004) and nonlantibiotics (reviewed by Eijsink et al., 2002), does not exist in the as-48 region analysed.

Gassericin A, reutericin 6 and acidocin B Biochemical and biological characteristics Gassericin A and reutericin 6 are two highly hydrophobic, circular bacteriocins produced by two species of LactobacilFEMS Microbiol Rev 32 (2008) 2–22

lus, Lactobacillus gasseri LA39 (Kawai et al., 1994) and Lactobacillus reuteri LA6 (Toba et al., 1991a, b), which were isolated within a 2-month interval from faeces of the same human infant. Both bacteriocins were thought to be the same substance because they have the same structural genes and cannot be distinguished by their molecular weights (MWs) or primary amino-acid sequences (Kawai et al., 2001). Mature bacteriocins (58-amino-acid residues, in which I1 and A58 must be covalently linked) were purified to homogeneity from culture supernatants by reverse-phase chromatography. They present more than 74% hydrophobic residues on their surfaces and are resistant to several peptidases and proteases, as are the other circular bacteriocins (Kawai et al., 1998, 2001). The MWs of both reutericin 6 and gassericin A, determined by time-of-flight mass spectroscopy, are 5652 and the only difference found between these bacteriocins is the presence of one (gassericin A) or two (reutericin 6) D-Ala residues in the molecule, which could be the cause of the differences in their modes of action and secondary structures (Kawai et al., 2004a). Bacteriocins with the same primary structures produced by different bacterial species have been reported for other lactic-acid bacteria (LAB) species such as Pediococcus acidilactici and Lactobacillus plantarum (pediocin AcH) (Motlagh et al., 1992; Ennahar et al., 1996) and Lactobacillus curvatus and Lactobacillus sake (curvacin A-sakacinA) (Axelsson & Holck, 1995), but the study by Kawai et al. (2004a) was the first to report the different characteristics of identical native primary bacteriocins such as gassericin A and reutericin 6, conferred by a difference in D-Ala contents in the molecules, apparently arising from an L-Ala to D-Ala conversion. The importance of the chirality of D-alanines in the activity of posttranslationally modified lantibiotics was studied by Skaugen et al. (1994). As has been demonstrated, the L-serines from lactocin S are enzymatically converted to D-Ala, giving rise to an apparent mistranslation of serine codons to alanine residue. It has been suggested that in such a case, this conversion results from a two-step reaction initiated by a lantibiotic synthetase converting the geneencoded L-serine to dehydroalanine (dha). Recently, Cotter et al. (2005b), using lacticin 3147 as a model system, identified the enzyme responsible for the conversion of dha to D-Ala, confirming the vital biological role for D-amino acids in the activity of this ribosomally synthesized peptide. As with the other bacteriocins analysed here, their secondary structures are organized mainly in a-helices (closer to the a/b type than the a1b), which yield a compact, stable structure and maintain their activity after having been heated to 100 1C for 60 min. Both bacteriocins have been described as acting in the cytoplasmic membrane of target cells and causing the death of the cell via an efflux of potassium ions, although they induce different quantities and patterns of this efflux. The activity of reutericin 6 is 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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influenced by substances on the surface of the indicator cells, which may cause the time lag in the mode of action and the low efflux pattern (Kawai et al., 2004). Nevertheless, their inhibition spectra against a number of Gram-positive, foodborne, pathogenic bacteria, such as Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus, but not against Gram-negative organisms, do not completely coincide, suggesting that the secondary structures of the two bacteriocins are slightly different (Itoh et al., 1995; Kawai et al., 2004b). Interestingly, gassericin A inhibits the growth of Lactobacillus reuteri LA6, but reutericin 6, which has a narrower spectrum, does not inhibit the growth of Lactobacillus gasseri LA39, and in addition loses o 50% of its activity at low pH values. Acidocin B (58 amino acids) is another hydrophobic class IV bacteriocin produced by Lactobacillus acidophilus M46, which is active against Listeria monocytogenes, Clostridium sporogenes, Brochothrix thermosphacta, Lactobacillus fermentum and Lactobacillus delbrueckii ssp. bulgaricus, but inactive against most other Lactobacillus species (Leer et al., 1995). According to in silico analysis, although this peptide was not identified as a circular protein (Leer et al., 1995) it might be considered to be a natural variant of gassericin A (Kawai et al., 2004a) because of its high sequence similarity (98%), only differing in one residue (M24 V). According to alignment with gassericin B, acidocin A should have a MW of 5620 [although the weight deduced by Kawai et al. (2004a) is 5750], all this in spite of its apparent value of 2400 on sodiumdodecyl sulphate polyacyrlamide gel electrophoresis due to its hydrophobic nature (Kawai et al., 2004b). Acidocin B is susceptible to trypsin, remains stable after heating to 80 1C for 20 min and can be extracted from the fluid culture supernatant with butanol (ten Brink et al., 1994). It has been established that growth is not necessary for its production because washed producer cells can synthesize the bacteriocin in a defined production medium. Identification of the gaaA and acdB structural genes The structural gene of gassericin A (gaaA), located on the chromosomal DNA of Lactobacillus gasseri LA39, was found as an ORF that encoded a protein of 91 amino acids with a calculated MW of 9268, which was sited downstream of another ORF encoding a 60-amino-acid protein that remains to be identified (Kawai et al., 1998) (GenBank accession number AB007043). Both ORFs were deduced to have a putative Shine–Dalgarno (SD) sequence, although no promoter-like sequence was identified upstream. More recently, however, a gene encoding a protein with a putative ATP-binding domain probably involved in the secretion of gassericin has been located downstream of gaaA (Kawai et al., 2004a). From the DNA sequence and results of mass 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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analysis, however, the amino-acid sequence of mature gassericin A was predicted to be 58 residues long, beginning with Ile as the N-terminal residue. The putative leader peptide sequence of this bacteriocin contains 33 residues with no Gly–Gly motifs but with a cleavage site (NH2 . . . TTANIYWIADQF . . . ) (Fig. 2) (Kawai et al., 1998). In a similar manner, the bacteriocin structural gene of reutericin was located on the chromosome of Lactobacillus reuteri LA6 (Kawai et al., 2004b). The genes encoding the production of acidocin B have been located on the pCV461 plasmid of Lactobacillus acidophilus M46 (GenBank accession number Z34920). DNA sequence analysis revealed the presence of three consecutive ORFs, which potentially encode hydrophobic peptides of 60, 91 and 114 amino acids, and a fourth one of opposite polarity, which may well encode a peptide of 59 amino acids (Leer et al., 1995) (Table 3 and Fig. 3). By comparing the amino-acid composition of highly purified acidocin B with the deduced amino-acid sequence, ORF-2 was identified as the structural gene (acdB) while the next gene encoded a protein with a putative ATP-binding domain involved in the secretion of gassericin A (Kawai et al., 2004b). Moreover, Leer et al. (1995) suggest the existence of a gene that encodes an immunity protein for one of the three nonassigned ORFs. Finally, analysis of the intergenic region carried out here with the MFOLD program revealed the existence of a long IR with an estimated DG free energy of 20.70 kcal mol 1 and the capacity to form a complex secondary structure (Fig. 4).

Table 3. Characteristics of predicted proteins specified by the gene cluster involved in the production of and immunity to gassericin A, acidocin B and butyrivibriocin AR10 (adapted from and Kawai et al., 2004a, b and Kalmokoff et al., 2003, respectively) Gene

Amino acid

Gassericin A gaaA 91 ORF1 60 ORF2 Acidocin B ORF1 60 acdB 91 ORF3 114 ORF4 59 Butyrivibriocin AR10 bviB 216 bviC 53 bviD 80 bviA 80 bviE ORF 6

Mass (kDa) 9.2 ND ND ND 9.2 ND ND 32.6 23.8 6.1 8.4

169

18.7

65

7.7

Function/homology Gassericin A precursor ATP-binding protein

Acidocin B precursor ATP-binding protein

ATP-binding protein (peripheral) Integral membrane protein (2 TM)? Membrane protein? Butyrivibriocin AR10 precursor / Gas A (38%) AcdB (47%) Membrane immunity protein/ ORF3 Acidocin B (29%) Peripheral?

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Butyrivibriocin AR10 Biochemical and biological characteristics Butyrivibriocin AR10, produced by the ruminal anaerobe Butyrivibrium fibrisolvens AR10, is a new member of the circular molecule group and shares a degree of homology with gassericin A (Kawai et al., 1998) and acidocin B (Leer et al., 1995). This bacteriocin has a wide spectrum of activity among Butyrivibrio isolates but its activity against other genera is fairly limited (Teather et al., 1999). Amino-acid analysis of butyrivibriocin AR10 purified to homogeneity confirmed a very high content of nonpolar residues, which is consistent with its extreme hydrophobicity, evident from both its elution characteristics with the reverse-phase column and the enhancement of activity in the presence of nonionic surfactants. As established by negative-ion, timeof-flight, mass-spectroscopic analysis, the MW of the isolated bacteriocin is 5981.5 Da, which corresponds to the molecular mass generated by the removal of the leader peptide and subsequent circularization, thus explaining the resistance to N-terminal Edman degradation (Kalmokoff & Teather, 1997). Its N-terminal sequence is homologous with that of gassericin A and both share features of sec-dependent leader peptidase cleavage sites (Kalmokoff et al., 2003). Moreover, the similarities between the N- and C-terminal regions of mature gassericin A before circularization (propeptide) and those predicted for butyrivibriocin AR10 (Fig. 2) would imply that they may be involved in the processing and cleavage events that go towards formation of the circular molecules, or else may be important for bacteriocin activity (reviewed by Kawai et al., 2004a). Functional DNA analysis of the bvi cluster genes A single sequence encoding a peptide of 80 amino acids was identified in the genome of Butyrivibrium fibrisolvens AR10 using a probe based on the amino-acid sequence of a cyanogen bromide (CNBr) fragment of the producer strain. Analysis in silico of the product of bviB, later renamed bviA (Kalmokoff et al., 2003), showed some sequence identity with gassericin A (38%) (Kawai et al., 1998) and acidocin B (47%) (Leer et al., 1995) (Table 3). The predicted aminoacid sequence of BviA is identical to the internal amino-acid sequence determined, except that found at position 18 of the internal peptide sequence (A18S), which has been interpreted as an amino-acid sequencing error (Kalmokoff et al., 2003). The predicted prepropeptide BviA (22 amino-acid residues) lacks the conserved double Gly leader peptidase processing motif common to the majority of previously characterized class II bacteriocins (Nes et al., 1996) but has a putative N-terminal cleavage site (NH2. . .LIPN YIFIADKM. . .) similar to that of the leader peptidase cleavage site in gassericin A (Kawai et al., 1998) (Fig. 2). FEMS Microbiol Rev 32 (2008) 2–22

Five nonoverlapping ORFs (bviBCDE and ORF 6) have been identified in the cloned region flanking the bacteriocin structural gene (Fig. 3) encoding peptides of 216, 53, 80, 169 and 64 amino acids (Table 3) (Kalmokoff et al., 2003) (GenBank accession number AF076529). Both Northern blotting and primer extension analysis indicate that the bviBCDAE region forms an independent transcriptional unit driven by a P1 promoter found 500-bp upstream of the start of bviB. Interestingly, the main transcript of 1 kb encompassed the bviA and bviE genes, although longer transcripts were also detected. This suggests the existence of a processing or degradation of a longer message encompassing the bviBCDAE genes. In the region preceding bviA, the presence of a small IR might explain the differences observed in the transcription levels of the bviBCD genes, present at relatively low levels compared with bviDE (Fig. 3) (Kalmokoff et al., 2003). Data concerning the enhanced levels of transcription of bviA in the presence of glucose, which increases culture density, suggest that butirivibriocin production may be regulated via a quorum-sensing mechanism, which would be in accordance with the existence of two ORFs (1 and 2) located immediately before the bvi operon and encoding a two-component regulator system, similar to those described in other bacteriocins (Fig. 3). Owing to the homology of BviB with the ATP-binding proteins and the predicted membrane-spanning domains of the hydrophobic proteins BviC and BviD (six and two TMs, respectively), it had been suggested that BviBCD might act as an ABC transport system involved in enhancing the production of and resistance to butyrivibriocin (Teather et al., 1999; Kalmokoff et al., 2003), while BviE has been considered to be the immunity protein (Kalmokoff et al., 2003).

Uberolysin Biochemical and biological characteristics Streptococcus uberis 42 produces two bacteriocins, a nisin U variant (Wirawan et al., 2006) and a second inhibitor, which has recently been identified as a novel member of the circular bacteriocins and named uberolysin (Table 1). This is an environmental microorganism belonging to the LAB that can be isolated from the lips and skin of cows, raw milk and udder tissue and is a major cause of bovine mastitis (Cullen, 1966; Khan et al., 2003). In contrast to other circular bacteriocins, uberolysin is an unusual 7048 Da protein because of its thermolability and capacity to lyse actively growing Gram-positive bacteria. Susceptible bacteria include most of the streptococci tested (except Streptococcus rattus and Streptococcus mutans), Listeria spp., enterococci, Micrococcus and Staphylococcus aureus (Wirawan et al., 2007). 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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As usual, the determination of the primary structure of uberolysin encountered an initial difficulty in that it was not susceptible to Edman degradation, suggesting that the Nterminal residue was blocked. For this reason, the authors analysed a linear fragment of 19 residues, spanning the Ala65 to Lys13 residues, which they obtained by tryptic digestion. MS/MS collision-induced fragmentation (CID) analyses confirmed that this fragment included the head-to-tail bond between the end residues from mature uberolysin and lacked further posttranslational modifications.

ducts of ublCD probably encode a transporter complex whereas UblB appears to be the equivalent of the CirB proteins (23% identity). The presence of an additional ABC-transporter has not, however, been detected in the uberolysin cluster but, as in the other bacteriocin clusters, a gene that may function as the putative immunity gene (ublE) has also been proposed.

Circularin A Biochemical and biological characteristics

Functional DNA analysis of the ubl cluster UblA is a 76 amino-acid polypeptide encoded by the ublA structural gene, having a short leader peptide consisting of only six amino acids, after the removal of which circularization occurs between L11 and W70 (Fig. 2). Sequencing of chromosomal DNA flanking the ublA gene revealed the existence of several ORFs, called ORF1, ublB, ublC, ublD and ublE (Table 4), which are probably involved in posttranslational modification, transport and immunity, and also possibly in the regulation of the biosynthetic gene cluster (GenBank accession number DQ650653). ORF1, located 303-bp upstream of ublA but in the opposite transcriptional direction (Fig. 3), encodes a ComE-like protein with conserved-domain homology to the LytR family of response regulators of the two-component signal transduction system. This type of regulator is involved in quorum sensing, biosynthesis of extracellular polysaccharides, fimbriation and expression of exoproteins, including toxins and bacteriocins (Nikolskaya & Galperin, 2002). It is also thought to be essential to uberolysin production, in spite of the absence of the corresponding ORF with homology to sensor kinases in its vicinity. The ubl cluster is in fact flanked (52 bp from each end) by two large 159 bp direct repeats (DR), and the authors wonder whether it may be involved in the acquisition of this gene cluster (Fig. 3). As usual, in the circular bacteriocins, the following ublBCDE genes, located 90 nt downstream of ublA, overlap in this intergenic region but an IR similar to that found in the majority of the cluster genes analysed here does not exist. Based on their amino-acid sequence similarities, the pro-

A new member of the circular bacteriocins, circularin A, a 69-residue peptide produced by Clostridium beijerinckii ATCC 25752, has been described by Kemperman et al. (2003a). Its inhibitory spectrum is quite narrow, although it inhibited all the Clostridium tyrobutyricum strains tested and also lactococci, enterococci and some Lactobacillus strains. Initial attempts to determine the N-terminal amino-acid sequence of the purified bacteriocin failed, indicating that the N terminus may be blocked. Its circular structure was resolved after purification to homogeneity from culture supernatants by reverse-phase chromatography and CNBr cleavage of the native protein into two fragments, which were then purified by HPLC to determine their amino-acid sequences (Kemperman et al., 2003b). Using BLAST searches against the NCBI nonredundant protein database or by direct comparison, Kemperman et al. (2003b) established a 60% homology with the amino-acid sequence of mature circularin A (69-amino-acid residue, pI 10.60) (30% identity) with that of AS-48, although it lacks the extended leader of this enterocin (Mart´ınez-Bueno et al., 1994; Kemperman et al., 2003b). In fact, only the first three N-terminal amino acids of pre-CirA (72 residues) are cleaved by hydrolysis between L 1 and V11, allowing the formation of a peptide bond between V11 and the Cterminal Y172 (Fig. 2b). Functional and transcriptional DNA analysis of the cir cluster The structural cirA gene was identified in the chromosome of Clostridium beijerinckii ATCC 25752 (GenBank accession

Table 4. Characteristic of predicted proteins specified by the ubl cluster from Streptococcus uberis 42, involved in the production of and immunity to uberolysin (adapted from Wirawan et al., 2007) ORF

Size (kDa)

pI

Homology

Hypothetical function

ORF1

Size (bp) 756

29.6

9.4

Response regulator

ublA ublB ublC ublD ublE

231 1608 546 657 267

7.8 63.5 21.3 25.5 10.2

8.5 9.5 8.7 4.8 9.2

Prepeptide CirB Integral membrane protein Cir D ATP-binding protein

Regulation of bacteriocin production Uberolysin precursor Circularization? ABC transporter ABC transporter Immunity protein

2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Genetics of circular bacteriocins

Table 5. Characteristics of predicted proteins specified by the cir gene cluster (adapted from Kemperman et al., 2003b) Gene

Size (bp)

Mass (kDa)

pI

Homology

cfgR cfgK cfg01 cfg02 cirA cirB cirC cirD cirE cirG cirH cirI

774 1275 597 390 216 1743 555 663 147 1425 744 1266

30.4 49.8 23.2 14.4 7.2 68.8 20.9 25.7 5.7 51.7 27.6 45.7

6 6.8 9.8 10.17 10.9 9.4 10.1 6.4 10.6 4.6 6.1 9.7

Response regulator Histidine kinase Hypothetical AgrB regulatory protein

Protein putative function

Enterocin AS-48 (30%) As-48B (19%) (11 TM) As-48C (21%) (4 TM) ATP-binding protein As-48D (31.6%) As-48D1 (30%) (2 TM) HlyD family (1TM)/As-48F (17%) ATP-binding protein As-48G (31.6%) Integral membrane (4 TM) As-48H (32%)

Cir A precursor Secretion/immunity Circularization? ABC transporter secretion/immunity Immunity protein Accesory protein ABC transporter ABC transporter ABC transporter permease

TM, transmembrane domains.

number AJ566621). The cirA gene – which is preceded by a Shine–Dalgarno sequence (AAGGAGGT) and a putative promoter region – encodes a peptide of 72 amino-acid residues, followed by a large palindromic structure (with an estimated DG free energy of 22.90 kcal mol 1) similar to that described for the majority of the structural genes reviewed (Fig. 4). In addition, an 11-kb region embracing the structural gene cirA encompasses two clusters of genes: the cir genes (cirABCDEGHI) involved in bacteriocin biosynthesis, maturation, secretion and immunity, and the cfg genes (cfgR, cfgK, cfg01, cfg02), two of which encode a twocomponent regulatory system. It is noteworthy that most of the gene products of the cir cluster show some degree of homology to the proteins involved in the production of AS48, although no homologue of the putative As-48C1 protein is found in the cir operon. Two modules of genes (cirABCDE and cirGHI) are putatively transcribed as polycistronic messengers (Fig. 3). The cirABCDE genes represent the minimum region required for bacteriocin biosynthesis, processing and extracellular production (Kemperman et al., 2003a): cirABCD gene products are required for the production of active circularin, while cirE seems clearly involved in bacteriocin immunity (Table 5), an assumption that will be discussed below. According to homology analysis, CirD and CirH both contain ATP-binding domains (Fig. 3) and CirG belongs to the HlyD family of accessory proteins of ABC transporters, while CirI, with four putative TMs and a Duf214 domain of predicted permeases, may be the membrane-spanning domain of a second ABC transporter (CirGIH). From the results obtained from single deletions in these genes, it has been concluded that CirB and CirD are required for partial resistance in the absence of CirE, but it seems clear that CirBD is principally involved in circularin secretion. It is worth noting, however, that the functional analysis proposed by Kemperman et al. (2003a) for the gene products CirB to CirI was performed mainly on the basis of homologies to proteins involved in the production of FEMS Microbiol Rev 32 (2008) 2–22

enterocin AS-48, because the roles of the Cir proteins were not investigated. There is no experimental evidence concerning the protein(s) involved in the processing and/or circularization of the CirA prepeptide, and the suggestion for an essential CirC protein involved in performing this function (Table 5), either alone or together with CirB and/or CirD, was only made on the basis of its homology with As48C, which has, however, recently been identified as an accessory immunity determinant (Ferna´ ndez et al., 2008). As in the other cluster genes in circular bacteriocins, the genetic organization of the cir region is somewhat compressed in the form of several overlapping genes. Overlapping between the end of one gene (cfgR, cirC, cirD, cirH and cirI) and the predicted start of the downstream gene suggests regulation of expression by translational coupling, a common feature throughout the cir region (Fig. 3). For cirH, translational coupling to cirG would be the only means of expression, as it lacks any obvious ribosome-binding site. As far as the cfg cluster is concerned, CfgR and CfgK are homologous to the response regulators and histidine kinases of twocomponent regulatory systems, which are often involved in the regulation of bacteriocin expression, and their genes are normally located near the bacteriocin operon. Unfortunately, there have been no further reports about the possible involvement of CfgR and CfgK in CirA expression.

Subtilosin A Biochemical and biological characteristics Subtilosin A (35 amino acids) is the only anionic, antimicrobial, circular peptide produced by the soil bacterium Bacillus subtilis. In the same way as several of the bacteriocins reviewed here, subtilosin A is highly resistant to endoproteinases, is very stable at high temperatures and defies complete sequence analysis by Edman degradation and mass spectral examination (Babasaki et al., 1985; Marx et al., 2001). It shows bactericidal activity against a diverse 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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range of Gram-positive and Gram-negative bacteria, aerobes and anaerobes, although it is less effective against capsulated forms of some Gram-negative bacteria (Shelburne et al., 2007). Heat shock increases its effectiveness against certain Gram-negative strains but protease has no effect on its activity (Shelburne et al., 2007). The widespread occurrence of subtilosin A reflects important physiological roles for this bacteriocin, which is produced at the end of exponential growth, particularly under stress conditions, and with specific functions during both the anaerobic and biofilm growth of Bacillus subtilis (Stein et al., 2004). Structural studies have shown that subtilosin A is posttranslationally modified in a singular way, placing it in a new type of lantibiotic, classified as class I (Table 1). According to the structure proposed by Babasaki et al. (1985), the removal of a short leader peptide (only eight amino-acid residues) leads to a peptide of 35 amino acids in length with a circular peptide backbone (Fig. 2). Subtilosin A contains several small amino-acid residues (seven Gly, five Ala), which are distributed throughout the whole molecule, thus allowing considerable flexibility, although as a consequence of its circular structure the conformational space of the backbone is highly restricted. NMR studies of labelled subtilosin A (Kawulka et al., 2003, 2004) (Fig. 1a) are consistent with its determined molecular mass (3399.7 Da), chemical reactivity and amino-acid composition. These studies also show that in addition to its circular structure, presubtilin undergoes several processing steps including proteolytic cleavage at the N11 and a new posttranslational modification, the oxidative linkage of cysteine sulphur to the a-carbon of another amino-acid residue, which has never been observed before in any ribosomally synthesized peptide (Kawulka et al., 2004). In fact, three cross-links are formed between the sulphurs of Cys13, Cys7 and Cys4 and the a-positions of Phe22, Thr28 and Phe31, respectively, with the loss of a hydrogen from each of these six residues. The thioether linkages between Cys and Ser/Thr are characteristic of lantibiotics (Kleerebezem, 2004), but the Cys–Phe linkage constitutes a new thioether type in this posttranslationally modified peptide. Finally, the circularization of the presubtilosin peptide involves covalent linking of the C-terminal G35 with the N-terminal N11 (Zheng et al., 1999) (Fig. 2). All these features confer high rigidity upon this molecule, the plausible antimicrobial activity mechanism of which seems to follow a receptor-binding and -mediated lipidperturbation model (Thennarasu et al., 2005). Solid-state NMR and fluorescence data have led to a model depicting the topology of subtilosin A embedded in the hydrophilic/ hydrophobic interface of the lipid bilayer (Fig. 1a). It has been proposed that subtilosin A may bind to lipid bilayers and orientate itself in such a way that the tryptophan-bearing edge of the molecule is buried within the hydrophobic core so as to disturb the positioning of both the phosphate head 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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group and the methylene units near the glycerol backbone. This binding results in a leakage of the aqueous contents of small unilamellar vesicles, albeit at higher peptide concentrations. Thennarasu et al. (2005) have recently investigated the type of interactions of subtilosin A with various model membranes and have come to the conclusion that it is modulated by the lipid composition of the target, the concentration used in the assay being an important factor. Functional and transcriptional analysis of genes from the sbo-alb cluster Current findings indicate that the production of mature subtilosin A requires the expression of eight (sboA-albABCDEFG) of the nine genes identified in the chromosome of the Bacillus subtilis producer strains (GenBank accession number AJ430547). These clustered genes are transcribed from a promoter residing upstream of the sboA structural gene (Fig. 2) and their products are involved in the posttranslational modification and processing of presubtilosin, secretion and also immunity (Zheng et al., 1999, 2000). The first four genes from the spo-alb operon are highly conserved in various subspecies of Bacillus subtilis (96–100% amino-acid identity) while the remaining ones are less conserved (83–88% identity). The putative coding sequences of the alb operon (Table 6) encode proteins that potentially function in the processing and export of peptides: the albA and albF products are critical for production while the albB, albC and albD products have a role in export and immunity. AlbA, involved in the posttranslational modification of presubtilosin (Zheng et al., 2000), was identified as a member of the MoaA/NifB/PqqE family (Menendez et al., 1995) and possesses two Cys clusters, located at an Fe–S centre, which serve as the active sites in reactions involving hydration or dehydration of substrate compounds. The polypeptides encoded by albC, -E, and -F show primary structural similarity to known proteins: AlbC probably functions in the export as an ABC transport complex, AlbE is a processing protease and AlbF is a member of a family of zinc endoproteinases that may perform critical modifications of the presubtilosin peptide. It has been demonstrated that AlbB, -C and -D are required for full immunity. There is another gene, sboX, which presumably encodes a bacteriocin-like precursor peptide bearing a Gly–Gly motif resembling type II prebacteriocin cleavage sites (Zheng et al., 2000). A 55-bp sequence that potentially encodes a region of RNA secondary structure was identified 93 bp upstream of the albA start codon, overlapping the C-terminal coding end of sboA (Zheng et al., 1999) (Fig. 4). It is similar to a factorindependent transcriptional termination sequence that may reduce transcriptional read-through into the alb genes, thereby ensuring that the alb products are generated in FEMS Microbiol Rev 32 (2008) 2–22

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Table 6. Characteristics of the sbo and alb genes involved in the production of and immunity to subtilosin A (adapted from Zheng et al., 2000) Gene

Size (bp)

Amino acid

Mass (kDa)

sboA

132

43

4.3

albA albB

1347 162

449 53

51.4 6.1

albC

720

240

27.2

albD

1311

436

49.5

albE albF albG sboX

1161 1281 702 153

399 427 234 50

44.6 48.9 26.3 5.8

Homology/protein putative function Subtilosin A precursor (pro-peptide) MoaA/NifB/PqqE family ABC transporter. Export and immunity ATP-binding cassette (ABC transporter). Export and immunity ABC transporter. Export and immunity Processing protease Zinc endoproteinases Membrane protein? Bacteriocin-like precursor peptide

smaller quantities compared with the SboA substrate peptide, as occurs in other similarly organized bacteriocin biosynthesis operons (Jack et al., 1995; Mart´ınez-Bueno et al., 1998; Qi et al., 1999; Kemperman et al., 2003a). The regulation of the expression of the sbo-alb operon is quite complex in that it is induced in late growth cultures apparently in response to starvation and also to oxygenlimited and anaerobic conditions (Nakano et al., 2000). In fact, it has been demonstrated that the transcriptional regulation of the sbo-alb operon is under dual control and involves the negative control of the global transition-state, transcriptional regulatory protein AbrB (a regulatory protein that controls the nutritional stress response and processes of cell differentiation in Bacillus subtilis) and the twocomponent regulatory proteins ResD and ResE, which could positively regulate sbo-alb transcription by repressing the abrB gene or indirectly affecting the concentration of AbrB and/or its activity (Nakano et al., 2000). Besides this, it is exciting to notice that the variance of this operon may be involved in an evolutionary relationship among different producer strains (Stein et al., 2004).

peptides. Their selectivity seems to be located in the Cterminal parts, the bacteriocin and immunity proteins being located on opposite sides of the cell membrane without any direct contact between the two molecules. Therefore, either the membrane itself or a specific membrane component seems to play a crucial role as a recognition intermediary between the bacteriocin and the immunity protein (Drider et al., 2006). In contrast to this, products concerned with immunity in bacteriocins other than those in class IIa have also been identified as membrane-interacting immunity proteins, some having a lipid modification that is thought to anchor the peptide to the cytoplasmic membrane (Ra et al., 1996; Sarik et al., 1996; Mart´ınez-Bueno et al., 1998; Drider et al., 2006). When expressed in susceptible cells, immunity proteins provide strong protection against attack by exogenous cognate bacteriocins (Drider et al., 2006). It has been proposed that the immunity protein could interact with the bacteriocin-induced pore and thereby blocks leakage, or alternatively, with a bacteriocin receptor that inhibits its action either by altering the receptor conformation or by hiding its bacteriocin-binding site (Drider et al., 2006). In spite of their importance, however, these proteins’ mode of action is still poorly understood. Furthermore, the success of their protective function often depends on the coexistence of complementary ABC systems having specific secretory/ immunity functions. This type of transporter has been described in bacteriocins that open pores in the cytoplasmic membrane, and their way of providing immunity could be by preventing them from reaching the high local densities required for pore formation, i.e. by keeping the bacteriocin concentration in the cytoplasmic membrane below the critical level necessary for pore formation, either by active transport of the mature molecules to the inside of the cell or by the active extrusion of bacteriocin molecules. These ABC transporters seem to represent the conservation during evolution of a wide resistance mechanism with secretion/ protection functions, probably interacting co-operatively with the immunity protein. It is obvious that co-operatively acting, multiple immunity systems provide a higher degree of immunity than does each system alone.

Immunity determinants for circular bacteriocins Bacteriocin producers are immune to their own bacteriocin, which results from the presence of a specific immunity protein. Genes encoding immunity determinants are usually situated within or close to related operons containing the genes required for bacteriocin biosynthesis. To date, at least 20 putative immunity proteins from the expressing strains have been identified from DNA sequences and subgrouped according to sequence similarities (Drider et al., 2006) in spite of there appearing to be little sequence homology among them. Products with functions related to immunity have been identified as small, highly charged, membrane-associated FEMS Microbiol Rev 32 (2008) 2–22

The function of as-48D1 plus as-48EFGH and a-48C products in AS-48 immunity The results published by Martı´nez-Bueno et al. (1998) confirm that As-48D1 (56 amino acids) affords some degree of bacteriocin resistance by itself and thus it has been identified as the immunity determinant. Judging by its amino-acid sequence, the as-48D1 immunity gene encodes a small (6.3 kDa) cationic hydrophobic protein with the structure of an integral cell-membrane protein rather than being weakly associated with the membrane. In fact, the predicted hydrophobic profile and helical wheel 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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representation of As-48D1 suggest the existence of two transmembrane segments with a typical a-helix conformation and a C-terminal hydrophilic region (unpublished results). More recently, D´ıaz et al. (2003) have proposed that the internal, and possibly regulated, operon as-48EFGH is related to higher resistance against exogenously added AS48 and indeed must be involved in self-protection against AS-48. The function of As-48EGH seems to be to enhance the secretion of AS-48 and resistance to exogenous enterocin, expelling it from the membrane into the surrounding medium, helped by the As-48F protein, which seems to be involved in accelerating the release of ADP from the ATPbinding protein As-48G (D´ıaz et al., 2003). Finally, the as-48C gene, which is co-ordinately expressed with the as-48ABC operon, has recently been identified as an auxiliary immunity determinant. It has been postulated that the bacteriocin and the immunity-protein encoding genes must be cotranscribed to ensure that the producer strain is not killed by its own bacteriocin. The posttranscriptional regulation of as-48ABC, recently identified by Ferna´ ndez et al. (2008), should provide producer cells with maximized production of functional AS-48 without deleterious effect, before the entire immunity machinery, as-48D1 and as48EFGH determinants, which are located in an independent operon, begins to work. The bviE gene confers butyrivibriocin AR10 immunity The direct linkage and relative abundance of the transcript encompassing bviDE has led Kalmokoff et al. (2003) to propose that BviE is the immunity protein. In addition, the hydrophobic character of BviE and the prediction of membrane-spanning domains, indicating an envelope location, are both consistent with the protective function surmised for this protein. The ublE plus ublCD secretory genes confer uberolysin immunity According to the results presented by Wirawan et al. (2007), the ubl cluster contains only one gene that may function as the putative immunity gene (ublE), because no additional ABC-transporter has been detected in the uberolysin locus. It has been suggested, however, that a supplementary protective mechanism could be provided by the UblCD transporter involved in secretory function, which may be capable of conferring low levels of immunity. The cirE and cirBD (? ) products confer circularin A immunity Reduced circularin A sensitivity could be acquired via two independent mechanisms, one of which is based on the 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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expression of cirE and the other on the combined expression of an ABC transporter. CirE is a small protein with a high isoelectric point and is organized into two predicted transmembrane helices, indicating that the protein is possibly located within the membrane, in a manner similar to As-48D1, the immunity protein of AS-48, and also other bacteriocin immunity proteins. According to the results presented by Kemperman et al. (2003a), mutation in either cirB or cirD together with deletion of cirE led to the loss of the circularin A-resistant phenotype, in a similar way to the results obtained in the as-48 cluster after mutation of their respective as-48C1 or as-48D homologous genes (Mart´ınez-Bueno et al., 1998). Nevertheless, it is plausible that the second system conferring CirA immunity would depend on the combined expression of CirE with CirGHI. This suggestion is in accordance with the proposed homology with the BacGHI proteins, identical to the as-48FGH multicomponent ABC transport system (Tomita et al., 1997; D´ıaz et al., 2003; Kemperman et al., 2003b).

albB plus albCD , with secretory functions, confer subtilosin immunity Zheng et al. (2000) proposed that the small 59-amino-acid hydrophobic peptide encoded by the albB gene plays a critical role in immunity because its alteration severely diminishes subtilosin self-protection. The small, hydrophobic AlbB is also required for maximum bacteriocin production, just as the other immunity peptides encoded by lantibiotic biosynthesis operons are. How these products function in conferring bacteriocin production and also selfprotection is still not known, but mutations in albC and, to a lesser extent, in albD reduce the cell’s immunity to subtilosin and the amount of active peptide produced. This is explicable because the product of albC is a member of the ABC family of transport proteins, which participate in the export of subtilosin and have been shown in several bacteriocins required for complete immunity to the specific peptide produced.

Unanswered questions regarding the machinery involved in processing and circularization Despite present progress in defining the biology of circular bacteriocins, the understanding of the enzymatic mechanisms involved in the circularization processes is still incomplete and lacking experimental evidence. Nonetheless, considerable attention is being directed towards identifying the posttranscriptional machinery and processing steps involved in the reaction(s). The function of the leader peptides, the method of leader cleavage and the steps involved in the circularization of the peptide remain FEMS Microbiol Rev 32 (2008) 2–22

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unknown. It is still not known, for example, whether the reactions to join their ends from linear cleaved AS-48 precursors are prompted by two different enzymes in almost simultaneous steps or by two reactions catalysed by the same enzyme. It has been confirmed, however, that all the circular structural genes are translated into precursor peptides and it is proposed that the production of mature linear peptides requires the removal of the N-terminal leader and the joining of the pro-peptide ends by a covalent peptide bond (Fig. 2). Intriguingly, the leaders examined so far are either extended (33 or 35 residues for gassericin A and AS-48, respectively) or have only a few amino-acid residues (three, six and eight for circularin A, uberolysin and subtilosin A, respectively) and lack any conserved sequences. Hegde & Bernstein (2006) have recently reported the complexity of the leader sequences of secreted proteins with cleavable N-terminal signal sequences that mediate their targeting to and translocation across bacterial cytoplasm membranes. They propose that signal sequences contain information that specifies the choice of targeting pathway, the efficiency of translocation, the timing of cleavage and even postcleavage functions, and play an important role in modulating protein biogenesis. Nevertheless, the absence of homology between the amino acids involved in joining the termini and the differences found in the leaders of these circular proteins remains a puzzle. Some of the many questions still to be answered are: Is there some relationship between the sites at which this process takes place? Are the cleavage and secretion coupled? What is the nature of the putative translocation intermediate? Is ATP required for folding at the outside of the membrane? Are there different mechanisms governing the circularization process, or might they use the same secretory pathways? Nevertheless, in the scientific literature reviewed there have been attempts to assign such functions to some of the coding products identified. For example, when Kawulka et al. (2003) arrived at their conclusions about the genetic machinery required for the production of mature subtilosin A, they proposed that the two processing peptidases AlbE and AlbF might be candidates for cutting the leader from the prepeptide and circularizing the linear product. In addition, the albA product, belonging to the radical SAM protein family, which is involved in the catalysis of several unusual reactions, would form the required Cys–Phe linkage described above (Sofia et al., 2001). As far as circularin A is concerned, there is no experimental evidence to identify the protein(s) involved in the processing and/or circularization of the CirA prepeptide, although Kemperman et al. (2003b) suggest that CirC protein could be the candidate to perform this function, either alone or together with the CirB and/or CirD proteins. A similar situation has been described for ublB, which has been proposed as the protein involved in the circularization of uberolysin (Wirawan et al., 2007). NeverFEMS Microbiol Rev 32 (2008) 2–22

theless, these suggestions are based exclusively on homologies shared with the genes encoding circularin A or enterocin AS-48. Interestingly, Mart´ınez-Bueno et al. (1998) have proposed that As-48B, alone or in association with the As-48C1D translocation complex, might open a pore specifically to facilitate the exit of AS-48 molecules. This hypothesis could imply that the ultimate activity of the putative multicomponent As-48BC1D proteins would be to remove the leader peptide and promote head-to-tail circularization concomitantly with the export of the molecule. Recent results show, however, that cleavage or circularization (or both) must be fulfilled by specific machinery from bacteria belonging to the Enterococcus genus (Ferna´ ndez et al., 2007), assuming that processing may require chromosomally derived auxiliary proteins similar to those described for the circularization of pilin in Agrobacterium tumefaciens (Eisenbrandt et al., 2000; Lai et al., 2002). An understanding of the precise mechanism underlying the processing of AS48 and its transport to the cell surface is still a main challenge because no product with the necessary specific peptidase activity and the ability to produce the circular AS48 backbone has yet been identified in the as-48 cluster. Speculations still exist about the requirement of either one or two enzymes involved in this process: e.g. an atypical transpeptidase that would catalyse the necessary double reaction or alternatively a signal peptidase involved in the proteolytic cleavage to remove the 35-amino-acid leader peptide, followed by a second enzyme linking the N-terminal M11 to the C-terminal W170. Even more, if a normal signal peptidase is used and cleaves a peptide bond upstream of the H 1 residue, a second enzyme would be needed to remove the propeptide N-terminal amino acid(s) in excess and link the N-terminal M11 with the C-terminal W70 (Mart´ınez-Bueno et al., 1994). Finally, it may be very interesting to confirm whether AS-48 can only be expressed by its natural host (Enterococcus species) or also by species from Clostridium, as has been observed in circularin A, the closest circular bacteriocins described so far. Nevertheless, in order to further our understanding of the mechanisms by which they function, the current level of knowledge about prebacteriocin processing needs to be extended to include the enzymes involved.

Concluding remarks This review is devoted to describing the genetic characteristics of a unique group of circular bacteriocins produced exclusively by Gram-positive bacteria. Particular attention is paid to the genetic description of those bacteriocins that have been characterized at the molecular level, highlighting the most recent developments concerning the identification of the genes involved in the production and maturation of these extremely special proteins and the immunity of their 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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producer bacteria. Whenever possible, the elaborate transcriptional regulation of the gene clusters involved in their expression is also explored. The genes of this new family of circular bacteriocins are chromosomally encoded, except those encoding acidocin B and enterocin AS-48, for which genes have been located on plasmids, with the exception of an AS-48 variant (AS-48RJ), produced by Enterococcus faecium, the as-48 gene cluster of which has been located on the bacterial chromosome (Abriouel et al., 2005). As far as the similarities and specificities of the gene composition of these clusters are concerned, they are organized into polygenic operons, a well-known strategy of bacterial genomes to facilitate their co-ordinated regulation. This is true of the nine genes of the sbo-alb operon (7 kb) from Bacillus subtilis (Zheng et al., 2000) and the five genes ublABCDE and bviBCDAE) from Streptococcus uberis and Butyrivibrium fibrisolvens – although a two-component regulatory system has been identified in an operon upstream of the bvi cluster – (Kalmokoff et al., 2003), and possibly also true of the tentative triple-gene gaa operon (Kawai et al., 1998, 2004b). No information is available about the transcription of the ubl cluster. In other cases, the genes are spread between two gene modules transcribing as polycistronic messengers, where one operon carries the essential genes and a second one carries genes for bacteriocin secretion and immunity, as is the case of the cirBCDE and cirGHI genes in the cir region (Kemperman et al., 2003a, b) and the as-48ABC and as48C1DD1EFGH operons from the as-48 cluster (Mart´ınezBueno et al., 1998; D´ıaz et al., 2003). Interestingly, these modules are quite compressed, with overlapping genes requiring translational coupling, an additional strategy that ensures the appropriate ratio of all their products (McCarthy & Gualerzi, 1990; van de Guchte et al., 1992). An analysis of available DNA sequences (as-48, acd, bvi, ubl, cir, sbo/alb) with the MFOLD program revealed the existence of a long IR with the potential to form a complex secondary structure after the respective structural genes (Fig. 4), the ubl and bvi clusters being the exceptions. The positioning of the IR has usually been seen as evidence that it is a transcriptional terminator, acting as an attenuator to ensure that the proteins encoded by the last genes are present in small, catalytic amounts while the peptide bacteriocin is produced in larger quantities (Mart´ınez-Bueno et al., 1998; Zheng et al., 1999). Ferna´ ndez et al. (2008) have recently proposed, however, that in the as-48 cluster this structure really represents specific sites for mRNA processing, allowing posttranscriptional processing and differential stability of the primary transcript to control the levels of the encoded proteins. In a similar way, it has been suggested that the differences observed in the transcript levels in the bvi cluster may be regulated at the level of mRNA stability (Teather et al., 1999). This processing uncouples transcription and 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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translation during the expression of polygenic operons, favouring differential mRNA decays, which provide a high level of mRNA for an efficient pre-protein product translation in comparison with a less abundant RNA substrate for the synchronized synthesis of accompanying polypeptides, thus ensuring the maintenance of an appropriate stoichiometry between them. Finally, there is an alternative way of controlling the differential production of proteins from the same operon, i.e. via the use of internal promoters such as those that putatively precede the as-48D1 and as-48EFGH immunity genes in the multigenic as-48C1DD1EFGH operon, thus ensuring the appropriate levels of immunity/ resistance determinants against the high concentrations of AS-48 produced (D´ıaz et al., 2003). It is known that most secreted and many membrane proteins contain extended, cleavable, N-terminal leader sequences that mediate their targeting to and translocation across the bacterial cytoplasmic membrane. Nevertheless, in the case of subtilosin, uberolysin and circularin, with three-, six- and eight-residue leader peptides, respectively, it is not likely that these sequences contain information to control their maturation/secretion. On checking the different secretion pathways of the bacteriocins analysed for this review, it was observed that there was no conserved Gly–Gly leader peptidase processing motif in any of the prebacteriocins investigated. Kawai et al. (1998) and Kalmokoff et al. (2003) suggest, in accordance with common features with secdependent peptidase (a positively charged residue at the N-terminus, a hydrophobic core region and cleavage following a neutral amino acid in position 1) and the absence of an ABC transporter, that acidocin B, gassericin A and butirivibriocyn AR10 are secreted via the prepeptide translocase (sec) pathway. Enterocin AS-48 is, however, secreted by the products of an ABC transporter identified as As-48C1D, despite a histidine residue found at position-1, which is very unusual for a signal peptide cleavage site (Mart´ınez-Bueno et al., 1994, 1998). On the basis of this hypothesis, Kemperman et al. (2003a, b), Wirawan et al. (2007) and Zheng et al. (2000) have proposed that CirBD, UblCD and AlbCD constitute putative ABC transporters for circularin, uberolysin and subtilosin, respectively, in spite of the fact that their three- six- and eight-residue leader peptides do not seem to contain any information to control their maturation/secretion. Permeation of the membrane, leading to cell death, has been the accepted mechanism for the action of a large number of membrane-lytic polypeptides (Jenssen et al., 2006). All these bacteriocins share common antimicrobial activity at the cytoplasmic membrane of Gram-positive bacteria (except for AS-48, which is also active against Gram-negative) through a transient pore formation that results in their depolarization, thus causing the death of the susceptible cell. Nevertheless, and contrary to the principal FEMS Microbiol Rev 32 (2008) 2–22

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mechanisms for the activity of hydrophobic and low-MW bacteriocins (Moll et al., 1999), gassericin A and reutericin A do not cause efflux of ATP through the cytoplasmic membrane of susceptible strains, indicating that neither of these bacteriocins causes cell lysis or the leakage of substances with MWs of more than 507 (ATP). The binding of subtilosin A to the lipid bilayer results, however, in the leakage of aqueous contents from small unilamellar vesicles, albeit at higher peptide concentrations. Interestingly, the mechanism proposed for the molecular function of AS-48 involves transition from a water-soluble dimer form (DF-I) to a membrane-bound dimer form (DF-II) at the membrane surface by a nonspecific interaction, which leads to the formation of nonselective pores and the free diffusion of low-MW solutes (Fig. 1b). Nevertheless, prolonged incubation with increased AS-48 concentrations has also been shown to cause fusion of the membranes and even more chaotic disorganization as secondary effects (Ga´ lvez et al., 1991). The high cost in energy involved in the production of circular bacteriocins, associated with the complex immunity mechanism for the cells that include the immunity gene, encoding an immunity protein to protect the bacteriocin producer from its own bacteriocin, and the other accessory proteins described above must also be emphasized. It is known that the expression of several genes must be involved in a high level of precursor peptide production and ATP hydrolysis for the bacteriocin-mediated secretion of neosynthesized bacteriocins and also for the second ABCtransporter-dependent immunity when it exists. Moreover, it is still not known whether the posttranslational circularization also has some ATP requirements. Clearly, exciting challenges lie ahead during the years to come.

Acknowledgements The authors’ investigation into enterocin AS-48 was sup´ General de Investigacion ´ ported by the Spanish Direccion Cient´ıfica y T´ecnica (Projects BI˙O 98-0908-C02-01, BIO2001-3237 and BIO2005-01544) and the Junta de Andaluc´ıa (PAI CVI 160). The authors are grateful to Dr Soler-Gonza´ lez and Dr Ram´ırez-Rodrigo of the Departments of Biochemistry and Molecular Biology at Granada University for their help in the prediction and functional assignment of the As-48B protein. The authors thank A.L. Tate for revising our English text.

References Abriouel H, Valdivia E, G´alvez A & Maqueda M (1998) Response of Salmonella choleraesuis LT2 spheroplasts and permeabilized cells to the bacteriocin AS-48. Appl Environ Microbiol 64: 4623–4626.

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Abriouel H, Maqueda M, G´alvez A, Mart´ınez-Bueno M & Valdivia E (2002) Inhibition of bacterial growth, enterotoxin production, and spore outgrowth in strains of Bacillus cereus by bacteriocin AS-48. Appl Environ Microbiol 68: 1473–1477. Abriouel H, Lucas R, Ben Omar N, Valdivia E, Maqueda M, Mart´ınez-Ca˜namero M & Ga´ lvez A (2005) Enterocin AS-48RJ: a variant of enterocin AS-48 chromosomally encoded by the food isolate Enterococcus faecium RJ16. Syst Appl Microbiol 28: 383–397. Ananou S, Valdivia E, Mart´ınez-Bueno M, G´alvez A & Maqueda M (2004) Effect of combined physico-chemical preservatives on enterocin AS-48 activity against the enterotoxigenic Staphylococcus aureus CECT 976 strain. J Appl Microbiol 97: 48–56. Ananou S, G´alvez A, Mart´ınez-Bueno M, Maqueda M & Valdivia E (2005) Synergistic effect on enterocin AS-48 in combination with outer membrane permeabilizing treatments against Escherichia coli O157: H7. J Appl Microbiol 99: 1364–1372. Axelsson L & Holck A (1995) The genes involved in production of and immunity to sakacin A, a bacteriocin from Lactobacillus sake Lb706. J Bacteriol 177: 2125–2137. Babasaki K, Takao T, Shimonishi Y & Kurahashi K (1985) Subtilosin A, a new antibiotic peptide produced by Bacillus subtilis 168: isolation, structural analysis, and biogenesis. J Biochem 98: 585–603. Berry C, O’Neil S, Ben-Dov E, Jones AF, Murphy L, Quail MA, Holden MTG, Harris D, Zaritsky A & Parkhill J (2002) Complete sequence and organization of pBtoxis, the toxincoding plasmid of Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 68: 5082–5095. Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3: 238–250. Cleveland J, Montville TJ, Nes IF & Chikindas ML (2001) Bacteriocins: safe, natural antimicrobials for food preservation. Int J Food Microbiol 71: 1–20. Cobos E, Filimonov VV, G´alvez A, Maqueda M, Valdivia E, Mart´ınez JC & Mateo PL (2001) AS-48: a circular protein with an extremely stable globular structure. FEBS Lett 505: 379–382. Cole AM, Wabg W, Waring AJ & Lehrer RI (2004) Retrocyclins: using past as prologue. Curr Prot Pept Sci 5: 365–372. Cotter PD, Hill C & Ross RP (2005a) Bacteriocins: developing innate immunity for food. Nat Rev Microbiol 3: 777–788. Cotter PD, O’Connor PM, Draper LA, Lawton EM, Deegan LH, Hill C & Ross RP (2005b) Post-translational conversion of L-serines to D-alanines is vital for optimal production and activity of the lantibiotic lacticin 3147. Proc Natl Acad Sci USA 102: 18584–18589. Craik DJ (2004) Preface. Curr Prot Pept Sci 5. Craik DJ (2006) Seamless proteins tie up their loose ends. Science 311: 1563–1564. Craik DJ, Daly NL, Saska I, Trabi M & Rosengren KJ (2003) Structures of naturally occurring circular proteins from bacteria. J Bacteriol 185: 4011–4021. Cullen GA (1966) The ecology of Streptococcus uberis. Br Vet J 122: 333–339.

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D´ıaz M, Valdivia E, Mart´ınez-Bueno M, Fern´andez M, SolerGonz´alez AS, Ram´ırez-Rodrigo H & Maqueda M (2003) Characterization of a new operon, as-48EFGH, from the as-48 gene cluster involved in immunity to enterocin AS-48. Appl Environ Microbiol 69: 1229–1236. Drider D, Fimland G, H´ehard Y, McMullen LM & Pr´evost H (2006) The continuing story of class IIa bacteriocins. Microbiol Mol Biol Rev 70: 564–582. Eijsink VH, Axelsson L, Diep DB, H˚avarstein LS, Holo H & Nes IF (2002) Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication. Antonie van Leeuwenhoek 81: 639–654. Eisenbrandt R, Kalkum M, Lurz R & Lanka E (2000) Maturation of IncP pilin precursors resembles the catalytic dyad-like mechanism of leader peptidases. J Bacteriol 182: 6751–6761. Ennahar S, Aoude-Werner D, Sorokine O, van Dorsselaer A, Bringel F, Hubert JC & Hasselmann C (1996) Production of pediocin AcH by Lactobacillus plantarum WHE 92 isolated from cheese. Appl Environ Microbiol 62: 4381–4387. Fern´andez M, Mart´ınez-Bueno M, Mart´ın MC, Valdivia E & Maqueda M (2007) Heterologous expression of enterocin AS48 in several strains of lactic-acid bacteria. J Appl Microbiol 102: 1350–1361. Fern´andez M, S´anchez-Hidalgo M, Garc´ıa-Quint´ans N, ´ Mart´ınez-Bueno M, Valdivia E, Lopez P & Maqueda M (2008) Processing of the as-48ABC RNA in AS-48 enterocin production by Enterococcus faecalis. J Bacteriol, in press. Folli C, Ramazzina I, Arcidiaco P, Stoppini M & Berni R (2003) Purification of bacteriocin AS-48 from an Enterococcus faecium strain and analysis of the gene cluster involved in its production. FEMS Microbiol Lett 221: 143–149. Franz CM, van Belkum MJ, Holzapfel WH, Abriouel H & G´alvez A (2007) Diversity of enterococcal bacteriocins and their grouping in a new classification scheme. FEMS Microbiol Rev 31: 293–310. G´alvez A, Maqueda M, Valdivia E, Quesada A & Montoya E (1986) Characterization and partial purification of a broad spectrum antibiotic AS-48 produced by Streptococcus faecalis. Can J Microbiol 32: 765–771. G´alvez A, Maqueda M, Mart´ınez-Bueno M & Valdivia E (1989a) Bactericidal and bacteriolytic action of peptide antibiotic AS-48 against Gram-positive and Gram-negative bacteria and other organisms. Res Microbiol 140: 57–68. G´alvez A, Valdivia E, Mart´ınez M & Maqueda M (1989b) Bactericidal action of peptide antibiotic AS-48 against Escherichia coli K-12. Can J Microbiol 35: 318–321. G´alvez A, Maqueda M, Mart´ınez-Bueno M & Valdivia E (1991) Permeation of bacterial cells, permeation of cytoplasmic and artificial membrane vesicles, and channel formation on lipid bilayers by peptide antibiotic AS-48. J Bacteriol 173: 886–892. Gonz´alez C, Langdon GM, Bruix M, G´alvez A, Valdivia E, Maqueda M & Rico M (2000) Bacteriocin AS-48, a cyclic polypeptide structurally and functionally related to mammalian NK-lysin. Proc Natl Acad Sci USA 97: 11221–11226.

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c

M. Maqueda et al.

Hancock REW & Diamond G (2000) The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol 8: 402–410. Hancock REW & Sahl H-G (2006) Antimicrobial and hostdefense peptides as new anti-infective therapeutic strategies. Nature Biotech 24: 1551–1557. Hegde RS & Bernstein HD (2006) The surprising complexity of signal sequences. Trends Biochem Sci 31: 563–571. Heng NCK & Tagg JR (2006) What’s in a name? Class distinction for bacteriocins. Nat Rev Microbiol 4, doi:10.1038/ nrmicro1273-c1. Heng NCK, Wescombe PA, Burton JP, Jack RW & Tagg JR (2007) The diversity of bacteriocins produced by Gram-positive bacteria. Bacteriocins – Ecology and Evolution (Riley MA & Chavan MA, eds), pp. 45–92. Springer, Heidelberg, Germany. Itoh T, Fujimoto Y, Kawai Y, Toba T & Saito T (1995) Inhibition of food-borne pathogenic bacteria by bacteriocins from Lactobacillus gasseri. Lett Appl Microbiol 21: 137–141. Jack R, Bierbaum B, Heidrich C & Sahl H-G (1995) The genetics of lantibiotic biosynthesis. Bioessays 17: 793–802. Jenssen H, Hamill P & Hancock REW (2006) Peptide antimicrobial agents. Clinic Microbiol Rev 19: 491–511. Jim´enez MA, Barrachi-Saccilott AC, Valdivia E, Maqueda M & Rico M (2005) Design, NMR characterization and activity of a 21-residue peptide fragment of bacteriocin AS-48 containing its putative membrane interacting region. J Pept Sci 11: 29–36. Joosten HM, Nu˜nez M, Devreese B, Van Beeumen J & Marugg JD (1996) Purification and characterization of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4. Appl Environ Microbiol 62: 4220–4223. Kalkum M, Eisenbrandt R & Lanka E (2004) Protein circlets as sex pilus subunits. Curr Prot Pept Sci 5: 417–424. Kalmokoff ML & Teather RM (1997) Isolation and characterization of a bacteriocin (Butyrivibriocin AR10) from the ruminal anaerobe Butyrivibrio fibrisolvens AR10: evidence in support of the widespread occurrence of bacteriocin-like activity among ruminal isolates of B. fibrisolvens. Appl Environ Microbiol 63: 394–402. Kalmokoff ML, Cyr TD, Hefford MA, Whitford MF & Teather RM (2003) Butyrivibriocin AR10, a new cyclic bacteriocin produced by the ruminal anaerobe Butyrivibrio fibrisolvens AR10: characterization of the gene and peptide. Can J Microbiol 49: 763–773. Kawai Y, Saito T, Toba T, Samant SK & Itoh T (1994) Isolation and characterization of a highly hydrophobic new bacteriocin (gassericin A) from Lactobacillus gasseri LA39. Biosci Biotechnol Biochem 58: 1218–1221. Kawai Y, Saito T, Kitazawa H & Itoh T (1998) Gassericin A; an uncommon cyclic bacteriocin produced by Lactobacillus gasseri LA39 linked at N- and C-terminal ends. Biosci Biotechnol Biochem 62: 2438–2440. Kawai Y, Ishii Y, Uemura K, Kitazawa H, Saito T & Itoh T (2001) Lactobacillus reuteri LA6 and Lactobacillus gasseri LA39 isolated from faeces of the same human infant produce identical cyclic bacteriocin. Food Microbiol 18: 407–415.

FEMS Microbiol Rev 32 (2008) 2–22

21

Genetics of circular bacteriocins

Kawai Y, Ishii Y, Arakawa K, Uemura K, Saitoh B, Nishimura J, Kitazawa H, Yamazaki Y, Tateno Y, Itoh T & Saito T (2004a) Structural and functional differences in two cyclic bacteriocins with the same sequences produced by lactobacilli. Appl Environ Microbiol 70: 2906–2911. Kawai Y, Kemperman R, Kok J & Saito T (2004b) The circular bacteriocins gassericin A and circularin A. Curr Prot Pept Sci 5: 393–398. Kawulka K, Sprules T, McKay RT, Mercier P, Diaper CM, Zuber P & Vederas JC (2003) Structure of subtilosin A, an antimicrobial peptide from Bacillus subtilis with unusual posttranslational modifications linking cysteine sulfurs to acarbons of phenylalanine and threonine. J Am Chem Soc 125: 4726–4727. Kawulka K, Sprules T, Diaper CM, Whittal RM, McKay RT, Mercier P, Zuber P & Vederas JC (2004) Structure of subtilosin A, a cyclic antimicrobial peptide from Bacillus subtilis with unusual sulfur to R-carbon cross-links: formation and reduction of R-thio-R-amino acid derivatives. Biochem 43: 3385–3395. Kemperman R, Kuipers A, Karsens H, Nauta A, Kuipers O & Kok J (2003a) Identification and characterization of two novel clostridial bacteriocins, circularin A and closticin 574. Appl Environ Microbiol 69: 1589–1597. Kemperman R, Jonker M, Nauta A, Kuipers OP & Kok J (2003b) Functional analysis of the gene cluster involved in production of the bacteriocin circularin A by Clostridium beijerinckii ATCC 25752. Appl Environ Microbiol 69: 5839–5848. Khan IU, Hassan AA, Abdulmawjood A, Lammler C, Wolter W & Zschock M (2003) Identification and epidemiological characterization of Streptococcus uberis isolated from bovine mastitis using conventional and molecular methods. J Vet Sci 4: 213–224. Klaenhammer TR (1988) Bacteriocins of lactic acid bacteria. Biochimie 70: 337–349. Kleerebezem M (2004) Quorum sensing control of lantibiotic production; nisin and subtilin autoregulate their own biosynthesis. Peptide 25: 1405–1414. Konisky J (1982) Colicins and other bacteriocins with established modes of action. Annu Rev Microbiol 36: 125–144. Lai EM, Eisenbrandt R, Kalkum M, Lanka E & Kado CI (2002) Biogenesis of T pili in Agrobacterium tumefaciens requires precise VirB2 propilin cleavage and cyclization. J Bacteriol 184: 327–330. Langdon GM, Bruix M, G´alvez A, Valdivia E, Maqueda M & Rico M (1998) Sequence-specific 1H assignment and secondary structure of the bacteriocin AS-48 cyclic peptide. J Biomol NMR 1: 173–175. Leer RJ, van der Vossen JM, van Giezen M, van Noort JM & Pouwels PH (1995) Genetic analysis of acidocin B, a novel bacteriocin produced by Lactobacillus acidophilus. Microbiol 141: 1629–1635. Maisnier-Patin S, Forni E & Richard J (1996) Purification, partial characterisation and mode of action of enteroccocin EFS2, an antilisterial bacteriocin produced by a strain of Enterococcus

FEMS Microbiol Rev 32 (2008) 2–22

faecalis isolated from a cheese. Int J Food Microbiol 30: 255–270. Maqueda M, G´alvez A, S´anchez-Barrena MJ, Gonz´alez C, Albert A, Rico M & Valdivia E (2004) Peptide AS-48: prototype of a new class of cyclic bacteriocins. Curr Prot Pept Sci 5: 399–416. Mart´ınez-Bueno M, G´alvez A, Valdivia E & Maqueda M (1990) A transferable plasmid associated with AS-48 production in Enterococcus faecalis. J Bacteriol 172: 2817–2818. Mart´ınez–Bueno M, Maqueda M, G´alvez A, Samyn B, Van Beeumen J, Coyette J & Valdivia E (1994) Determination of the gene sequence and molecular structure of the enterococcal peptide antibiotic AS–48. J Bacteriol 176: 6334–6339. Mart´ınez-Bueno M, Valdivia E, G´alvez A, Coyette J & Maqueda M (1998) Analysis of the gene cluster involved in production and immunity of the peptide antibiotic AS-48 in Enterococcus faecalis. Mol Microbiol 27: 347–358. Marx R, Stein T, Entian K-D & Glaser SJ (2001) Structure of the Bacillus subtilis peptide antibiotic subtilosin a determined by 1H-NMR and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. J Prot Chem 20: 501–506. Mendoza F, Maqueda M, G´alvez A, Mart´ınez-Bueno M & Valdivia E (1999) Antilisterial activity of peptide AS-48 and study of changes induced in the cell envelope properties of an AS-48-adapted strain of Listeria monocytogenes. Appl Environ Microbiol 65: 618–625. Menendez C, Igloi G, Henninger H & Brandsch R (1995) A pAO1-encoded molybdopterin cofactor gene (moaA) of Arthrobacter nicotinovorans: characterization and site-directed mutagenesis of the encoded protein. Arch Microbiol 164: 142–151. McCarthy JE & Gualerzi C (1990) Translational control of prokaryotic gene expression. Trends Genet 6: 78–85. Moll GN, Konings W & Driessen A (1999) Bacteriocins: mechanism of membrane insertion and pore formation. Antonie van Leewenhoek 76: 185–198. Moreno F, San Millan JL, Hern´andez-Chico C & Kolter R (1995) Microcins. Biotechnology Series 28, Genetics and Biochemistry of Antibiotics Production (Vining LC & Stuttard C, eds), pp. 307–321. Butterworth-Heinemann, London. Motlagh AM, Bhunia AK, Szostek F, Hansen TR, Johnson MC & Ray B (1992) Nucleotide and amino acid sequence of pap-gene (pediocin AcH production) in Pediococcus acidilactici H. Lett Appl Microbiol 15: 45–48. Nakano M, Zheng G & Zuber P (2000) Dual control of sbo-alb operon expression by the Spo0 and ResDE systems of signal transduction under anaerobic conditions in Bacillus subtilis. J Bacteriol 182: 3274–3277. Nes IF, Diep DB, Havarstein LS, Brurberg MB, Eijsink V & Holo H (1996) Biosynthesis of bacteriocins in lactic acid bacteria. Antonie van Leeuwenhoek 70: 113–128. Nes IF, Diep DB & Holo H (2007) Bacteriocin diversity in Streptococcus and Enterococcus. J Bacteriol 189: 1189–1198. Nikolskaya A & Galperin MY (2002) A novel type of conserved DNA-binding domail in the transcriptional regulators of the AlR/AgrA/LytR family. Nucleic Acids Res 30: 2453–2459.

2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

c

22

Nissen-Meyer J & Nes IF (1997) Ribosomally synthesized antimicrobial peptides: their function, structure, biogenesis and mechanism of action. Arch Microbiol 167: 67–77. O’Connor EM & Shand RF (2002) Halocins and sulfolobicins: the emerging story of archaeal protein and peptide antibiotics. J Ind Microbiol Biotech 28: 23–31. Qi F, Chen P & Caufield PW (1999) Functional analyses of the promoters in the lantibiotic mutacin II biosynthetic locus in Streptococcus mutans. Appl Environ Microbiol 65: 652–658. Ra SR, Wiao M, Immonen T, Pujana I & Saris EJ (1996) Genes responsible for nisin synthesis, regulation and immunity form a regulon of two operons and are induced by nisin in Lactococcus lactis N8. Microbiol 142: 1281–1288. Riley MA & Wertz JE (2002) Bacteriocin diversity: ecological and evolutionary perspectives. Biochimie 84: 357–364. Ross RP, Morgan S & Hill C (2002) Preservation and fermentation: past, present and future. Int J Food Microbiol 79: 3–16. Samyn B, Mart´ınez-Bueno M, Devreese B, Maqueda M, G´alvez A, Valdivia E, Coyette J & Van Beeumen J (1994) The cyclic structure of the enterococal peptide antibiotic AS-48. FEBS Lett 352: 87–90. S´anchez-Barrena M, Mart´ınez-Ripoll G, G´alvez A, Valdivia E, Maqueda M, Cruz V & Albert A (2003) Structure of bacteriocin AS-48: from soluble state to membrane bound state. J Mol Biol 334: 541–549. Sarik PE, Immonen T, Reis M & Sahl HG (1996) Immunity to lantibiotics. Antonie van Leeuwenhoek 69: 151–159. Selsted M (2004) y-defensins: cyclic antimicrobial peptides produced by binary ligation of truncated a-defensins. Curr Prot Pept Sci 5: 365–372. Shelburne ChE, An FY, Dholpe V, Ramamoorthy A, Lopatin DE & Lantz MS (2007) The spectrum of antimicrobial activity of the bacteriocin subtilosin A. J Antimicrob Chemother 59: 297–300. Skaugen M, Nissen-Meyer J, Jung G, Stevanovic S, Sletten K, Inger C, Abildgaard M & Nes IF (1994) In vivo conversion of Lserine to D-alanine in a ribosomally synthesized polypeptide. J Biol Chem 269: 27183–27185. Sofia HJ, Chen G, Ketzler BG, Reyes-Spindola JF & Miller NE (2001) Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucleic Acids Res 29: 1097–1106. Stein T, D¨usterhus S, Stroh A & Entian K-D (2004) Subtilosin production by two Bacillus subtilis subspecies and variance of the sbo-alb cluster. Appl Environ Microbiol 70: 2349–2353.

2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

c

M. Maqueda et al.

Teather RM, Kalmokoff ML & Whitford MF (1999) The role of bacteriocins in rumen microbial ecology. The Rumen Microbial Ecosystem. Microbial Biosystems: New Frontiers. Proceedings of the 8th International Symposium on Microbial Ecology (Bell CR, Brylinsky M & Johnson-Green P, eds) Atlantic Canada Society for Microbial Ecology, Halifax, Canada. ten Brink B, Minekus M, van der Vossen JM, Lee RJ & Huis in’t Veld JH (1994) Antimicrobial activity of lactobacilli: preliminary characterization and optimization of production of acidocin B, a novel bacteriocin produced by Lactobacillus acidophilus M46. J Appl Bacteriol 77: 140–148. Thennarasu S, Lee D-K, Poon A, Kawulka KE, Vederas JC & Ramamoorthy A (2005) Membrane permeabilization, orientation, and antimicrobial mechanism of subtilosin A. Chemist Phys Lipids 137: 38–51. Toba T, Samant SK, Yoshioka E & Itoh T (1991a) Reutericin 6, a new bacteriocin produced by Lactobacillus reuteri LA6. Lett Appl Microbiol 13: 281–286. Toba T, Yoshioka E & Itoh T (1991b) Potential of Lactobacillus gasseri isolated from infant faeces to produce bacteriocin. Lett Appl Microbiol 12: 228–231. Tomita H, Fujimoto S, Tanimoto K & Ike Y (1997) Cloning and genetic and sequence analyses of the bacteriocin 21 determinant encoded on the Enterococcus faecalis pheromoneresponsive conjugative plasmid pPD1. J Bacteriol 179: 7843–7855. Trabi M & Craik DJ (2002) Circular proteins-no end in sight. Trends Biochem Sci 27: 132–138. van de Guchte M, Kok J & Venema G (1992) Gene expression in Lactococcus lactis. FEMS Microbiol Rev 88: 73–92. Wirawan RE, Klesse NA, Jack RW & Tagg JR (2006) Molecular and genetic characterization of a novel nisin variant produced by Streptococcus uberis. Appl Environ Microbiol 72: 1148–1156. Wirawan RU, Swanson KM, Kleffmann T, Jack RW & Tagg JR (2007) Uberolysin: a novel cyclic bacteriocin produced by Streptococcus uberis. Microbiol 153: 1619–1630. Zheng G, Yan LZ, Vederas JC & Zuber P (1999) Genes of the sboalb locus of Bacillus subtilis are required for production of the antilisterial bacteriocin subtilosin. J Bacteriol 181: 7346–7355. Zheng G, Hehn R & Uber P (2000) Mutational analysis of the sboalb locus of Bacillus subtilis: identification of genes required for subtilosin production and immunity. J Bacteriol 182: 3266–3273. Zuker M & Mathews DH (1999) Turner algorithms and thermodynamics for RNA secondary structure prediction. A Practical Guide in RNA Biochemistry and Biotechnology. NATO ASI Series (Barciszewski J & Clark BFC, eds), pp. 11–43. Kluwer Academic Publishers, Dordrecht.

FEMS Microbiol Rev 32 (2008) 2–22