Bacteriocin Diversity in Streptococcus and Enterococcus�
Ingolf F. Nes,* Dzung B. Diep, and Helge HoloLaboratory of Microbial Gene Technology, Department for Chemistry, Biotechnology and Food Science,
Norwegian University of Life Sciences, N1432 Ås, Norway
Most bacteriocins in gram-positive bacteria are small andheat stable (peptide bacteriocins), and their antimicrobial ac-tivities are directed against a broader spectrum of bacteriathan is seen for bacteriocins of gram-negative bacteria. Manyexcellent bacteriocin reviews have been published in recentyears (10, 15, 16, 19, 27, 29, 77, 83).
The heat-stable peptide bacteriocins from lactic acid bac-teria have so far been grouped into two major classes: classI, the lantibiotics, and class II, the heat-stable nonlantibiot-ics. In addition, a third class of bacteriocins has been sug-gested which includes secreted heat-labile cell wall-degrad-ing enzymes (71, 88), but classification of such enzymes asbacteriocins has recently been disputed (19, 49). Lantibiot-ics contain a number of posttranslational modifications thatinclude dehydration of serine and threonine to form 2,3-dehydroalanine (Dha) and 2,3-dehydrobutyrine (Dhb), re-spectively. Some of the dehydrated residues are covalentlybound to the sulfur in neighboring cysteines, creating thecharacteristic lantionine and methyllantionine residues. Ithas also been shown that in a few cases the dehydroalaninecan be converted to D-alanine (109, 118) and that additionalmodifications, such as lysinoalanine, 2-oxobutyrate, S-ami-novinyl-D-cysteine, and S-aminovinyl-D-methylcysteine, areformed in some lantibiotics (59). Both class I and class IIbacteriocins display great diversity with regard to theirmodes of action, structures, genetics, modes of secretion,choices of target organisms, etc. There is still lack of con-sensus on how to subdivide class I and II peptide bacterio-cins further into subclasses. The lantibiotics have been di-vided into two subgroups, type A and type B, according tostructural features (64). Type A lantibiotics (e.g., nisin, sub-tilin, and Pep5) are elongated molecules with a flexiblestructure in solution, while type B lantibiotics adapt a morerigid and globular structure (64). However, this picture ischanging, since structural studies of the lantibiotic planta-ricin C has been shown to hold structural elements of bothtype A and B lantibiotics (123). Also, nuclear magneticresonance spectroscopy has shown that the peptides of thetwo-peptide lantibiotic lacticin 3247 are structurally differ-ent. While the peptide designated lacticin 3147 A1 has aspecific lanthionine bridging pattern resembling the globu-
lar type B lantibiotic mersacidin, the A2 peptide is a mem-ber of the elongated type A lantibiotic subclass (80). In thepresent review, we refer to the A and B types of lantibioticsas one-peptide lantibiotics and mention specifically when abacteriocin is a two-peptide lantibiotic.
Lack of consensus also exists in the differentiation betweensubgroups of the nonlantibiotic class II peptide bacteriocins. Inthis review, we retain the pediocin-like bacteriocin in class IIa,the two-peptide bacteriocins in class IIb, and the leaderlesspeptide bacteriocins in class IIc, and finally, we define thecircular bacteriocins as class IId. This overview will discuss thedissemination of the class I and II peptide bacteriocins inenterococci and streptococci and the possibility of identifyingsuch bacteriocins in genome sequences.
The lactic acid bacteria in fermented food have been thefocus of bacteriocin research during the last 15 to 20 years.Numerous peptide bacteriocins have been characterized, andmany have been used intentionally or unintentionally in foodproducts, either through starter cultures or as food additives/supplements (15). Work on such bacteriocins has been drivenby the need for new and improved natural food preservationtechnology and a wish to prevent food spoilage and poison-producing bacteria from growing. In recent years, the conceptof probiotic bacteria has also stimulated work on bacteriocins.In light of the increased antibiotic resistance among pathogens,bacteriocins have attracted attention as an alternative means toprevent infection by pathogens. In fact, two lantibiotics, nisinand lacticin 3147, have been found useful in preventing mas-titis (8, 108, 110).
The nonfood lactic acid bacteria, such as enterococci andstreptococci, have also been scrutinized for bacteriocin pro-duction, and many publications have shown that they are pro-ducers of such antimicrobial peptides (Tables 1 and 2). Someenterococci are actually part of the main fermenting flora ofartisan dairy and meat products, particularly in Mediterraneancountries. Their presence in the fermented products is mostprobably doe to the relatively high temperatures found in thatregion (35).
The enterococci are among the dominant lactic acid bac-teria in the intestinal flora of mammals and other animals.The streptococci constitute part of the complex oral micro-flora of mammals, and some of them are pathogenic. Studiesof the antimicrobial peptides from streptococci and entero-cocci have shown that these bacteria produce a large num-ber and diversity of such compounds. It should be noted thatidentical peptide bacteriocins have been isolated by differ-ent research groups, and unfortunately, they have been
* Corresponding author. Mailing address: Laboratory of MicrobialGene Technology, Norwegian University of Life Sciences, P.O. Box5003, N-1432 Ås, Norway. Phone: 47 64965878. Fax: 47 64941465.E-mail: [email protected].
given different names. In addition, many bacteriocins havebeen only partly characterized, and available information isoften insufficient to confirm their novelty. The present re-view covers only the best-defined class I and class II bacte-riocins from enterococci and streptococci.
DIVERSITY AND DISSEMINATION OFPEPTIDE BACTERIOCINS
Streptococcus. Studies of the bacteriocins of streptococci goback to the 1960s (84). The more recent focus in the isolationand characterization of streptococcal bacteriocins has been onpathogenic streptococci, and most bacteriocins characterizedoriginate from a few species (Table 1). Lantibiotics are themost prevalent peptide bacteriocins in streptococci, and themajority belong to the elongated cationic type A lantibiotics.Two-peptide lantibiotics have also been isolated from strepto-cocci (Table 1).
The lantibiotic salivaricin A was the first Streptococcus sali-varius lantibiotic to be characterized (107), and it stronglyinhibits Streptococcus pyogenes strains. S. pyogenes (a group AStreptococcus) is a ubiquitous organism that is known to pro-voke a wide variety of diseases in humans. Five additionalvariants of the structural salivaricin A (salivaricin A1 to A5)peptide have been identified in streptococcal strains, and to-gether with salivaricin A, these six SalA peptides seem to sharean inhibition spectrum, probably because they differ from eachother only by 1 or 2 amino acids (128). Among the SalA-typepeptide bacteriocins, SalA1 activity was most prevalent, beingproduced by a broad range of species, including S. pyogenes,Streptococcus dysgalactiae, and Streptococcus agalactica.Among the 53 different M types of S. pyogenes strains, all butone carried the salA1 gene variant; however, none was found tobe a bacteriocin producer. On the other hand, 77% of 36 S.
TABLE 1. Peptide bacteriocins isolated from streptococci
Organism Bacteriocin Type Mass (Da)(amino acids) Reference(s)
S. salivarius Salivaricin A Class I 2,315 (22) 107, 119, 128S. salivarius Salivaricin B Class I 2,733 (25) 119S. salivarius Salivaricin A2 Class I 2,364 (22) 119S. pyogenes Streptococcin A-FF22 Class I 2,795 (26) 55, 60S. macedonicus Macedocin Class I 2,795 40S. pyogenes Streptin Class I 2,424 (23) 68, 127S. mutans Mutacin I, mutacin II (H-29B) Class I, class I 2,364 (24) 3,245 (27) 11, 73, 92, 101S. mutans Mutacin N 4,806 (49) 4S. mutans Mutacin B-Ny266 Class I 2,270 (22) 86S. mutans Mutacin III (Mutacin1140) Class I 2,263 (22) 50, 102S. bovis Bovicin HJ50 Class I 3,428 (33) 133S. uberis Nisin U Class I nisin-like 3,029 (32) 132S. mutans SmbA, SmbB Class I two-peptide lantibiotic (30, 32) 135S. rattus BHT-A(Smb-like); BHT-Aa, BHT-Ab Class I Two-peptide lantibiotic 2,802 (30), 3,375 (32) 54S. mutans Mutacin IV; peptide A, peptide B Class II two-peptide bacteriocin 4,169 (44), 4,826 (49) 100S. thermophilus Thermophilin 13 (A); ThmA, ThmB Class II two-peptide bacteriocin 5,776 (62), 3,910 (43) 78S. bovis Bovicin 255 Class II 5,968 (56) 129S. rattus BHT-B Class II nonleader 5,195 (44) 54
TABLE 2. Peptide-bacteriocins isolated from enterococci
Organism Bacteriocin Type Mass (Da)(amino acids) Reference(s)
E. faecalis Cytolysin Cyl�L, Cyl�S Class I two-peptide lantibiotics 3,458 (38), 2,032 (21) 56E. faecium Enterocin A Class IIa pediocin-like 4,829 (47) 3, 93, 96E. faecium Enterocin P Class IIa pediocin-like 4,493 (44) 12E. faecium Bac 32 Class IIa pediocin-like 7,998 (70) 58E. faecium Bacteriocin GM-1 Class IIa pediocin-like 4,630 (44) 67E. faecalis Bac 31 Class IIa pediocin-like (43) 58E. mundtii Mundticin ATO6, mundticin KS, enterocin
salivarius strains were positive for the salA gene, as well asproducing the bacteriocin (128).
Streptolysin S (SLS), which is responsible for the hallmarkbeta-hemolytic phenotype, is produced by group A Streptococ-cus. A nine-gene locus is necessary and sufficient for its bio-synthesis, and the genes resemble genes required for the syn-thesis of some bacteriocins. Although SLS does not antagonizebacteria, the biosynthesis elements needed for its synthesissupport the designation of SLS as a bacteriocin-like toxin (37,53, 95).
Peptide bacteriocins produced by Streptococcus mutans areknown as mutacins, and the frequency of such antimicrobialactivities has been reported to vary from 11% to 100% amongS. mutans isolates (5). A study of mutacin production in 145oral S. mutans isolates from young children and their mothersshowed that 88% of the strains produced antimicrobial activityagainst more than 1 of the 14 indicator strains (42).
Some S. mutans isolates produce at least three differentlantibiotics named mutacin I, II, and III. Mutacin II has beenshown to be structurally related to the lacticin 481 group oftype A lantibiotics (73), while the structures of mutacins I andIII have not been determined. A two-peptide class II peptidebacteriocin (mutacin IV) is produced by S. mutans UA140, astrain that also produces mutacin I (100). The production ofmutacins I and IV by UA140 appears to be regulated by dif-ferent mechanisms under different physiological conditions inthe sense that mutacin I is only produced by cells growingunder biofilm-forming conditions, while mutacin IV is pro-duced in planktonic cultures. The production of two mutacinsby one strain under different conditions implies that they servedifferent roles in the ecology of S. mutans (100).
The evolution and dissemination of such peptide bacterio-cins in lactic acid bacteria were recently demonstrated by nisin,the most prominent lantibiotic, so far found only in Lactococ-cus lactis strains. A recent publication by Wirawan and cowork-ers described a nisin variant called nisin U (78% identity tonisin A) from Streptococcus uberis (132). This bacterium isprimarily found on the lips and skin of cows, in raw milk, andon udder tissue and is a major cause of bovine mastitis (116,136, 137).
Two bacteriocins, termed bovicin 255 and bovicin HJ50,have been thoroughly characterized from a rumen bacterialisolate of Streptococcus bovis (129, 133). Bovicin HJ50 is alantibiotic, while bovicin 255 is a class II nonlantibiotic. In thesame study, 7 out of 35 rumen streptococcal isolates werefound to produce bacteriocin-like activity (129). The broaddissemination of bovicin 255 among rumen streptococci hasrecently been confirmed (18). The bovicin 255 peptide se-quence showed some similarity to those of two other class IIpeptide bacteriocins, lactococcin A (51) and thermophilin A(thermophilin 13) (125), and it was identified in both Strepto-coccus gallolyticus and S. bovis isolates.
In another study, a Streptococcus rattus isolate produced twodifferent peptide bacteriocins named BHT-A and BHT-B. Thegenetic loci for the BHT-A and BHT-B bacteriocins werefound in six S. rattus and two S. mutans strains (54). BHT-Awas found to be a variant of the two-peptide lantibiotic Smb(135), while BHT-B was a nonmodified peptide with somesimilarity to the tryptophan-rich and leaderless aureocin A53from Staphylococcus aureus (54, 91).
In a screening of Streptococcus pyogenes, approximately 10%of the strains were found to inhibit the growth of nine indica-tors in a standardized streptococcal-bacteriocin assay (127).The bacteriocin activity was due to the type A lantibiotic strep-tin, and two major forms of streptin were purified to homoge-neity from an S. pyogenes strain (127). The fully matured formof streptin (streptin 1) is made up of 23 amino acid residuesand has a mass of 2,424 Da, while the second form of thepeptide (streptin 2) has three additional amino acids (TPY) atthe N terminus. The structural gene (strA) of streptin waswidespread among S. pyogenes strains. Of 58 S. pyogenes iso-lates tested, 41 hybridized with the strA probe, but only 10 ofthe strains produced active streptin. The deficiency of somestrains in producing streptin was ascribed to a deletion in theirstreptin loci, encompassing genes putatively encoding proteinsinvolved in streptin processing (68, 127). The strA gene wasfound to be absent in S. salivarius (75 isolates), S. mutans (8isolates), and S. uberis (9 isolates).
Streptococcal bacteriocins have also been characterizedfrom food-related environments, such as food fermentation.Macedocin is a lantibiotic from the newly described speciesStreptococcus macedonicus, isolated from artisan cheese (40), andthermophilin 13 is a two-peptide class IIb bacteriocin produced byStreptococcus thermophilus, isolated from yogurt (78).
Peptide bacteriocin activities have been described in manymore streptococcal isolates, but often the characterization isincomplete, which makes it difficult to judge if they are newbacteriocins. It is surprising to see the high frequency of pep-tide bacteriocin-producing streptococcal isolates, and this ob-servation is supported by gene annotation, as well as genomemining of sequenced Streptococcus genomes (see below).
Enterococcus. Many peptide bacteriocins from enterococcihave been purified and genetically characterized over theyears, and most of them have been obtained from Enterococcusfaecalis and Enterococcus faecium. The best characterized onesare listed in Table 2. As observed in streptococci, identicalcharacterized peptide bacteriocins have been given differentnames, such as bacteriocin AS-48, which has been named bothenterocin 4 (62) and bacteriocin 21 (121).
The bacteriocins from enterococci belong almost exclusivelyto the heat-stable, nonlantibiotic class II bacteriocins, with theexception of cytolysin. Cytolysin is a two-peptide lantibioticfound in E. faecalis (17). It is the only peptide bacteriocinisolated from enterococci with cytolytic (hemolytic) activity(56, 61, 70). Cytolysin is a virulence factor, and consequently,it is not considered useful as an antimicrobial agent. It isinteresting that the cytolysin-encoding genes are found notonly in Enterococcus isolates from hospitals and patients, butalso from food; animals, including houseflies; and healthy in-fants (65, 76, 115).
Probably the most prevalent class II enterococcal bacterio-cins are pediocin-like bacteriocins (class IIa) with strong anti-listerial effects (30, 31). Among these bacteriocins, enterocin Awas the first to be thoroughly characterized, and this peptidebacteriocin is among those most frequently found in E. faeciumstrains (3, 122). The gene for enterocin A has also been iden-tified in the partly sequenced genome of E. faecium strain DO,but the strain does not produce any bacteriocin activity (http://genome.jgi-psf.org/draft_microbes/entfa/entfa.home.html).While most class IIa bacteriocins are processed from a precur-
sor peptide containing an N-terminal double-glycine leadersequence (between 15 and 30 amino acid residues), some pep-tide bacteriocins, such as enterocin P and bacteriocin 31, con-tain a sec-like leader sequence that is removed in the secretionprocess (12, 122).
The two-peptide peptide bacteriocins (class IIb) require thecomplementary actions of both peptides for full antimicrobialactivity, and only one immunity protein is dedicated to theactivity of a two-peptide bacteriocin. In some E. faecalisstrains, the two-peptide bacteriocin named enterocin 1071 isencoded by plasmid-derived genes (34). The two peptides,enterocin 1071A and enterocin 1071B, constituting the bacte-riocin are 39 and 34 amino acids long, respectively. The de-duced amino acid sequences of the mature Ent1071A andEnt1071B peptides showed 64 and 61% homology with the �and � peptides of lactococcin G, respectively, a two-peptidebacteriocin isolated from L. lactis (94).
Several leaderless peptide bacteriocins have been identifiedamong enterococci (Table 2). The leaderless peptide bacterio-cins were first discovered in staphylococci and were shown tobe hemolytic (26, 90, 91, 126); they are produced as singlepeptides or multiple homologous peptides, each encoded byindividual genes localized in tandem repeats (91). The multi-ple-peptide bacteriocins (up to four individual peptides)shared 65 to 80% homology (14, 26, 91, 126). From the foodisolate E. faecium strain L50, two plasmid-encoded leaderlessbacteriocins were characterized. One, termed enterocin L50,was composed of two highly similar peptides sharing 72%identity. The individual peptides enterocin L50A and enterocinL50B possessed antimicrobial activity, with the L50A peptidebeing most active. However, in combination, the antimicrobialactivity increased between 5- and 80-fold, depending on thebacterial indicator used (14). The enterocin L50-encodinggenes were localized as two consecutive open reading frames(ORFs) on a 50-kb plasmid (20). The second leaderless pep-tide bacteriocin, enterocin Q, was isolated from the same E.faecium strain. Enterocin Q consists of only one peptide, andits gene was found on 7,383-bp plasmid (pCIZ2). Recently, twoleaderless bacteriocin peptides (MR10A and MR10B) werecharacterized from an E. faecalis strain isolated from the ho-locrine glands of a bird (81). The two MR10 peptides werealmost identical to the enterocin L50 peptides.
It is interesting that some leaderless peptide bacteriocins inenterococci share homology with Staphylococcus peptides thatappear to be hemolytic (14). An intriguing question is, cansome few amino acid substitutions render the L50 peptidescytolytic? As previously mentioned, the enterococcal lantibi-otic cytolysin, as well as the SLUSH peptides (26) and the AGSpeptides (126), most likely encompass both cytolytic and bac-tericidal activities, which suggests that such activities can co-exist in the same peptide molecule.
A unique group of peptide bacteriocins, classified as classIId in this review, are the cyclic bacteriocins. One member ofthis group, enterocin AS-48, has been identified in E. faecalis.Enterocin AS-48 was the first bacteriocin isolated from Entero-coccus to be purified (38, 82). Screening by PCR-based tech-nology of 15 independently isolated bacteriocin-producingEnterococcus strains for the structural genes similar to theantimicrobial peptide AS-48 gene has been carried out (63).Eight of 10 E. faecalis strains and 3 of 5 E. faecium strains gave
positive results. This finding suggests that peptide bacteriocinsclosely related or identical to peptide AS-48 are common inenterococci (63). The almost identical cyclic enterocin AS-48RJ has been characterized from an E. faecium strain. Itdeviates from enterocin AS-48 only in amino acid position 20,replacing glutamine with valine (1).
In which ecological environments are bacteriocin-producingenterococci commonly found? Presently, there does not seemto be any preferential niche; wherever one finds enterococci,one also finds bacteriocin-producing enterococci. A major por-tion of the bacteriocin-producing enterococci have been iso-lated from foods (cheese, meat, fish, and vegetables), animals,and humans (33). A collection of 636 hospital-isolated vanco-mycin-resistant E. faecium strains were tested for bacteriocinproduction. It was shown that 44% of the strains were bacte-riocin producers, and a significant number of these strainscarried the genetic determinant for bacteriocin 32 production(58). Bacteriocin-producing enterococci have also been iso-lated from municipal sewage, cattle dung, ruminal content,birds, etc. (58, 74, 75, 79).
Production of multiple bacteriocins also seems to be a com-mon feature of enterococci and streptococci. It has been re-ported that many isolates produce three or four bacteriocins(13, 98, 131).
PEPTIDE BACTERIOCIN GENES INGENOME SEQUENCES
Bacterial genome sequences are revolutionizing our ap-proach to identifying novel genes in different bacteria. Morethan 300 complete bacterial genome sequences are available inpublic-domain databases, and the number is increasing. Suchsequence databases should be an extremely valuable source inwhich to look for genes encoding antimicrobial compounds,such as ribosomally synthesized antimicrobial peptide bacte-riocins (89). The peptide bacteriocins are small molecules thatare consequently encoded by small genes, and unfortunately,small ORFs are often not adequately annotated or are omitted inannotation. There is a need for improved search tools for iden-tification of peptide bacteriocin genes, and there should be morefocus on the annotation of small ORFs. In the case of Streptoco-coccus genomes, numerous putative peptide bacteriocin-encodinggenes have been proposed. In the genome sequence of Strepto-coccus pneumoniae TIGR 4, seven ORFs have been annotated asbacteriocin genes, while four bacteriocin genes have been anno-tated in the S. pneumoniae R6 genome (http://cmr.tigr.org/tigr-scripts/CMR/CmrHomePage.cgi).
Results from an in silico screening for peptides containingthe double-glycine leader sequence and their cognate trans-porter have been published (25). The double-glycine leadermotif that is found in a majority of class II and some class Ibacteriocins and the unique N-terminal peptidase C39 domainof their cognate ABC transporters were the main features usedin this search for such peptides (45, 46). The study included 45fully sequenced gram-positive genomes, and of a total of 48GG motif candidate peptides obtained, 92% were found inlactic acid bacteria and 80% were found in the streptococcalgenomes. However, one should bear in mind that peptidepheromones (including competence peptides) involved in thetwo-component regulatory systems are also detected in such a
search (87). The peptide pheromones share most features ofbacteriocins but are usually shorter and have no or poor anti-microbial activity (24).
A more general and probably more efficient peptide bacte-riocin search engine has been developed (21). The genome-mining tool includes a search for lantibiotics, as well as peptidebacteriocins with different N-terminal leaders (21). This web-based genome-mining tool (with the acronym BAGEL) appliesa number of ORF prediction tools that take into account thepresence of genes involved in biosynthesis machinery, trans-port function, regulation, and immunity. These features makeBAGEL unique, as well as valuable, in searches for putativebacteriocin genes and their biosynthetic operons in bacterialgenomes (21).
In the annotated Streptococcus genome sequences, a numberof new peptide bacteriocin genes have been proposed. In the S.pneumoniae TIGR 4 genome sequence, seven putative peptidebacteriocin genes have been annotated (SP0042, SP0109,SP0531, SP0532, SP0533, SP0539, and SP0541), but no bacte-riocin activity has been characterized so far (120). Evaluationof the peptide-bacteriocin genes in S. pneumoniae TIGR 4 byuse of the BAGEL algorithm (default setting) identified 11significant peptide bacteriocin genes, including the seven orig-inal putative bacteriocin genes and four additional genes(SP974, SP0540, SP792, and SP602). The program also iden-tified 18 other potential bacteriocin genes and a high numberof ORFs (44) with some, but less, homology to bacteriocingenes. A recent published work explored the distribution ofgenes in eight clinical isolates of S. pneumoniae by constructingan individual genomic library for each isolate. DNA sequenc-ing suggested that one isolate contained a gene similar to theglobular lantibiotic mersacidin, probably in two copies (117).
Peptide bacteriocin genes have been identified in other se-quenced streptococcal species. S. mutans UA159, a sequencedcariogenic dental pathogen, exhibits nonlantibiotic mutacin ac-tivity (2). Several putative bacteriocin ORFs in S. mutansUA159 share strong homology with ORFs found in genomes ofS. bovis and S. pneumoniae strains. The annotation of S. mu-tans UA159 originally suggested six hypothetical peptide bac-teriocin ORFs, one of which (the translated protein Q8CVC8)showed homology with bovicin 255 variants and acidocin M(66). A seventh translated ORF (the protein Q8DS95) mayalso encode a bacteriocin, since it shares homology with aputative bacteriocin of S. thermophilus (gi 62528196). In a re-cent study, it was demonstrated by bioinformatics and muta-tional analyses that the antimicrobial repertoires of S. mutansstrain UA159 includes the two-peptide mutacin IV (SMU 150and SMU151) and mutacin V (SMU1914c) (44).
Streptococcin A-FF22 and the streptins (streptin 1 and 2)are the only peptide bacteriocins thoroughly characterizedfrom S. pyogenes strains (55, 60, 127). The genome sequencesof seven S. pyogenes strains (MGAS1039, MGAS315,MGAS5005, MGAS6180, MGAS8232, SF370, and SSI-1) arelisted in the TIGR-CMR database, and more than 30 ORFsencoding bacteriocin-like proteins have been annotated, withprevalences varying from two to seven putative bacteriocinORFs in each strain.
Enterococci produce many different peptide bacteriocins,and purification and characterization have shown that the non-lantibiotic peptide bacteriocins dominate among them. The
genome sequence of E. faecalis V583, a vancomycin-resistantclinical isolate, has been published, but it is the only completeEnterococcus genome sequence available so far (99). No puta-tive bacteriocin ORFs were initially identified, but a hypothet-ical protein of 43 amino acids (EFA0015) is almost identical tothe recently characterized plasmid-encoded leaderless entero-cin EJ97 (112). The translated peptide EFA0015 is 97% iden-tical to enterocin EJ97, as it lacks the threonine in position 14of enterocin EF97. However, it remains to be seen if theprotein EFA0015 is an antimicrobial compound.
It must be emphasized that identification of putative bacte-riocin genes does not necessarily mean that the relevant bac-terium produces antimicrobial activity, and a lack of detectableantimicrobial activity does not necessarily mean that genesinvolved in bacteriocin production are defective. First, it is ofkey importance to use a susceptible indicator, which can pose aproblem, since some peptide bacteriocins act on only a narrowrange of target bacteria. Secondly, the production of peptidebacteriocins is often regulated. The best-understood regulatorysystem for peptide bacteriocin production is the two-componentsystem (87), but other regulatory mechanisms also exist (13, 104,105). Deficiency in production of antimicrobial activity is oftendue to a dysfunctional genetic system. Occasionally, it has beenobserved that bacterial genomes encode only parts of the bacte-riocin production system or that mutations have inactivated thefunctionality of the bacteriocin genes (23, 85).
REGULATION OF PEPTIDE BACTERIOCIN SYNTHESIS
Bacteriocins may play important roles in bacterial ecology,and the high incidence of bacteriocin production among strep-tococci and enterococci probably reflects this fact. In mostcases, bacteriocin production appears to be regulated and isconsequently produced only under suitable growth conditions.Therefore, the choice of the right culturing conditions (me-dium composition, temperature, pH, water activity, etc.) maybe crucial for the outcome of bacteriocin screenings.
Some bacteriocins are produced on solid growth media butnot in liquid cultures (100). Growth temperatures have alsobeen shown to influence bacteriocin production in E. faecium. E.faecium L50 produces at least three bacteriocins, and it was dem-onstrated that the various bacteriocins were produced at differenttemperatures and had different temperatures for optimal produc-tion (13). For the production of bacteriocins from S. pyogenes, thepresence of blood was essential, and the same requirement wasalso reported for the cytolysin of E. faecalis (17).
Bacteriocin production has been shown to be regulated bytwo-component systems in a number of lactic acid bacteria,including S. thermophilus (52) and E. faecium (93). In suchcases, the inducers are highly specific autoinduced bacteriocin-like peptides that are commonly referred to as peptide pher-omones (87). The regulatory circuit of enterocin A and B wasamong the first to be elucidated for enterococci; the individualgenes involved, the sequence of the peptide pheromone, andthe promoter sequences are shown in Fig. 1 (93). The induc-tion of gene expression is achieved by an accumulation of thepheromone peptide through a low constitutive production.When a threshold concentration of the peptide pheromone hasbeen reached, the peptides binds to its receptor (the histidineprotein kinase), followed by a phosphorylation cascade leading
to phosphorylation of the cognate response regulator, whichbinds and activates the regulated promoters (Fig. 1). A burst inthe expression of genes takes place, and mass production ofbacteriocin results (87).
In E. faecium CTC492, enterocin A production was dimin-ished at low pH and high concentrations of salt but was re-stored by the addition of inducer peptide (93). In E. faecalis,one of the two peptides of cytolysin, CylLS� peptide, induceshigh-level expression of the cytolysin structural genes (57).However, the second component, CylLL� peptide, can form acomplex with CylLS that prevents induction. CylLL binds pref-erentially to target cells (erythrocytes), and in the presence oftarget cells, high-level cytolysin expression is induced. Thus,this autoregulatory mechanism provides the bacteria with ameans to fine tune cytolysin production in response to thepresence of targets (17).
TARGETS—RECEPTORS FOR BACTERIOCINS
Numerous mode-of-action studies have been performed onpeptide bacteriocins. Most bacteriocins are membrane active,causing permeabilization of and eventually killing the targetbacteria. Both some A- and B-type lantibiotics have beenshown to kill target cells by interrupting cell wall synthesisthrough high-affinity binding to the lipid II molecule, a mole-cule that plays an essential role in the synthesis of the pepti-doglycan layer (6, 7, 97). Type A lantibiotics are also able to killbacteria by an additional mechanism: binding to the lipid IImolecule and thereby forming pores in the cytoplasmic mem-brane of the target. The mechanisms of pore formation of typeA lantibiotics are the most important killing mechanism. Asimilar pore formation mechanism has also been shown for thetwo-peptide lantibiotic lacticin 3147 (130). At present, we donot know the details of the mechanisms of action of lantibiotics
from streptococci, but it seems likely that some of these lan-tibiotics, such as mutacin I, 1140, and B-Ny266, also use lipid IIas a target molecule (10).
Targets for class II bacteriocins are less well known. How-ever, genetic studies have suggested that the mannose PTSsystem is the target of class IIa bacteriocins (47). Based onthese genetic studies and biochemical studies (D. B. Diep andH. Holo, unpublished data), a model of how class IIa bacte-riocins work and how the dedicated immunity protein canprovoke its activity has been developed (Fig. 2).
CONCLUDING REMARKS
Enterococci and streptococci seem to be unique in theirgreat potential to produce peptide bacteriocins. It is interestingthat while lantibiotics are by far the peptide bacteriocins mostfrequently found in streptococci, the class II peptide bacterio-cins are dominant in enterococci. Most of the purified strep-tococcal bacteriocins are plasmid encoded, but bacterial chro-mosomally encoded bacteriocins have also been isolated, andgenome mining suggests the presence of a large number ofbacteriocin genes in their genomes. In spite of the great num-ber of putative bacteriocin genes in the sequenced S. pneu-moniae strains, no bacteriocin activity has been reported. Thisis in contrast to S. mutans, from which many bacteriocins havebeen purified and characterized and for which coordinatedbacteriocin production and competence development havealso been observed (72, 124). In the early 1970s, it was reportedthat bacteriocin synthesis coincided with DNA uptake compe-tence development in Streptococcus gordonii strain Challis(113, 114), and recently, it was reported that bacteriocin/he-molysin biosynthesis was controlled by the competence regulon(48). These findings suggest that bacteriocins play an impor-
FIG. 1. Genomic organization of the enterocin A and B loci (3, 9, 36, 93, 96). (A) Genes involved in production of enterocin A and enterocinB in Enterococcus faecium. The enterocin A locus consists of two operons: (i) the bacteriocin operon consisting of the enterocin A gene (entA),the immunity gene of EntA (entI), the peptide pheromone gene (entF), the receptor of the peptide pheromone, the histidine protein kinease gene(entK), and the DNA binding activator, the response regulator (entR), and (ii) the second operon (transporter operon) consisting of the two genes,the ABC transporter (entT) and its accessory gene (entD), that are needed for the secretion of both the peptide pheromone and the bacteriocin.The enterocin B locus consists of two divergent operons: (i) the monocistronic operon consisting of the enterocin B gene (entB) and (ii) the secondoperon, containing the immunity gene (eniB). Both operons are controlled by the regulatory genes (entFKR) of enterocin A, and the processingand transport of enterocin B are probably mediated by the entT and entD genes. The four regulated promoters are indicated by arrows. ORFs ofunknown function are shown as open arrows. (B) DNA sequences of the regulated promoter regions. The direct-repeat sequences that are thebinding sites for the phosphorylated response regulator are in boldface and underlined. Putative �35 and �10 regions are shown in boldface italics.(C) Deduced precursor of the peptide pheromone (induction peptide) EntF. The sequence of the mature peptide pheromone is shown in boldface.
tant role in providing naked DNA for uptake in competentstreptococci.
The lack of direct correlation between the many putativepeptide bacteriocin genes in the Streptococcus strains and an-timicrobial activity expressed is puzzling. The possibility cannotbe excluded that the lack of activity of such translated ORFs issimply because they are not antimicrobial peptides but serve acompletely different and unknown function. In this context,one should bear in mind that peptide pheromones for compe-tence development, as well as regulation of bacteriocin pro-duction in gram-positive bacteria (quorum sensing), sharemany of the physiochemical properties of bacteriocins (87). Inorder to determine if an ORF may encode a peptide bacteri-ocin, different strategies should be considered. Heterologousexpression of peptide bacteriocins has been achieved, but suchan approach depends on the presence of complementary genesinvolved in maturation and transport, in addition to immunity(106). Peptide synthesis is an alternative strategy that has beenused successfully to obtain class II peptide bacteriocins (32),while chemical synthesis of lantibiotics cannot presently beobtained due to the extensive posttranslational modifications.
Correction of a mutation in a gene required for bacteriocinsynthesis has been used successfully to identify a new bacteri-ocin in lactic acid bacteria (23). The genome of Pediococcuspentosaceus ATCC 25745 contains a gene cluster that resem-bles a regulated bacteriocin system. A mutated and defectivepeptide pheromone involved in a quorum-sensing regulatorymechanism for bacteriocin synthesis was identified. Geneticcorrection of the mutated peptide pheromone made it possibleto express the bacteriocin.
Enterococci produce a great diversity of class II peptidebacteriocins, and most of them are plasmid encoded. Unfor-tunately, only one Enterococcus genome has been sequencedcompletely, and only one putative bacteriocin gene was iden-tified. From this limited information, it is not possible to drawany firm conclusions about the prevalence of bacteriocin-en-coding traits in enterococcal chromosomes. However, we canconclude that enterococci produce a great number of differentclass II bacteriocins and that most of them are apparentlyplasmid encoded. It is tempting to speculate that bacteriocinsare found more frequently in enterococci and streptococci thanin many other lactic acid bacteria, such as Lactococcus andLactobacillus. Genome mining suggests that there is great po-tential to find many new bacteriocins in Streptococcus, and itwill be important to follow up such findings with functionalstudies, which hopefully will bring new and efficient antimicro-bial peptides to the market in the future.
ACKNOWLEDGMENT
We thank the Norwegian Research Council for funding.
REFERENCES
1. Abriouel, H., R. Lucas, N. Ben Omar, E. Valdivia, M. Maqueda, M.Martinez-Canamero, and A. Galvez. 2005. Enterocin AS-48RJ: a variant ofenterocin AS-48 chromosomally encoded by Enterococcus faecium RJ16isolated from food. Syst. Appl. Microbiol. 28:383–397.
2. Ajdic, D., W. M. McShan, R. E. McLaughlin, G. Savic, J. Chang, M. B.Carson, C. Primeaux, R. Tian, S. Kenton, H. Jia, S. Lin, Y. Qian, S. Li, H.Zhu, F. Najar, H. Lai, J. White, B. A. Roe, and J. J. Ferretti. 2002. Genomesequence of Streptococcus mutans UA159, a cariogenic dental pathogen.Proc. Natl. Acad. Sci. USA 99:14434–14439.
3. Aymerich, T., H. Holo, L. S. Havarstein, M. Hugas, M. Garriga, and I. F.Nes. 1996. Biochemical and genetic characterization of enterocin A fromEnterococcus faecium, a new antilisterial bacteriocin in the pediocin familyof bacteriocins. Appl. Environ. Microbiol. 62:1676–1682.
4. Balakrishnan, M., R. S. Simmonds, A. Carne, and J. R. Tagg. 2000. Strep-tococcus mutans strain N produces a novel low molecular mass non-lantibi-otic bacteriocin. FEMS Microbiol. Lett. 83:165–169.
5. Balakrishnan, M., R. S. Simmonds, M. Kilian, and J. R. Tagg. 2002.Different bacteriocin activities of Streptococcus mutans reflect distinct phy-logenetic lineages. J. Med. Microbiol. 51:941–948.
6. Bonelli, R. R., T. Schneider, H. G. Sahl, and I. Wiedemann. 2006. Insightsinto in vivo activities of lantibiotics from gallidermin and epidermin mode-of-action studies. Antimicrob. Agents Chemother. 50:1449–1457.
7. Breukink, E., and B. de Kruijff. 2006. Lipid II as a target for antibiotics.Nat. Rev. Drug. Discov. 5:321–332.
8. Broadbent, J. R., Y. C. Chou, K. Gillies, and J. K. Kondo. 1989. Nisininhibits several gram-positive, mastitis-causing pathogens. J. Dairy Sci. 72:3342–3345.
9. Casaus, P., T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernandez, and H. Holo.1997. Enterocin B, a new bacteriocin from Enterococcus faecium T136which can act synergistically with enterocin A. Microbiology 143:2287–2294.
10. Chatterjee, C., M. Paul, L. Xie, and W. A. van der Donk. 2005. Biosynthesisand mode of action of lantibiotics. Chem. Rev. 105:633–684.
11. Chikindas, M. L., J. Novak, A. J. Driessen, W. N. Konings, K. M. Schilling,and P. W. Caufield. 1995. Mutacin II, a bactericidal antibiotic from Strep-tococcus mutans. Antimicrob. Agents Chemother. 39:2656–2660.
12. Cintas, L. M., P. Casaus, L. S. Havarstein, P. E. Hernandez, and I. F. Nes.1997. Biochemical and genetic characterization of enterocin P, a novelsec-dependent bacteriocin from Enterococcus faecium P13 with a broadantimicrobial spectrum. Appl. Environ. Microbiol. 63:4321–4330.
13. Cintas, L. M., P. Casaus, C. Herranz, L. S. Havarstein, H. Holo, P. E.Hernandez, and I. F. Nes. 2000. Biochemical and genetic evidence thatEnterococcus faecium L50 produces enterocins L50A and L50B, the sec-dependent enterocin P, and a novel bacteriocin secreted without an N-terminal extension termed enterocin Q. J. Bacteriol. 182:6806–6814.
14. Cintas, L. M., P. Casaus, H. Holo, P. E. Hernandez, I. F. Nes, and L. S.Havarstein. 1998. Enterocins L50A and L50B, two novel bacteriocins fromEnterococcus faecium L50, are related to staphylococcal hemolysins. J.Bacteriol. 180:1988–1994.
15. Cleveland, J., T. J. Montville, I. F. Nes, and M. L. Chikindas. 2001. Bac-teriocins: safe, natural antimicrobials for food preservation. Int. J. FoodMicrobiol. 71:1–20.
16. Coburn, P. S., and M. S. Gilmore. 2003. The Enterococcus faecalis cytolysin:
FIG. 2. Model of the mechanisms behind killing (A) and immunity(B) of classIIa bacteriocins. (A) The bacteriocin (red) employs man-PTS (orange) as a target receptor upon approaching susceptible cells(1). It binds to the components IIC (C) and IID (D) of mannose-PTS(2) and somehow causes leakage of solutes across the cytoplasmicmembrane (3) and eventually cell death. (B) In immune and non-bacteriocin-producing cells (1), the immunity protein (pink) is nonas-sociated or loosely associated with the receptor proteins. When bac-teriocin is exogenously added or produced by the bacteria themselves(2), the immunity protein is tightly associated with the receptor toprevent the bound bacteriocin on the receptor from forming lethalpores in the cytoplasmic membrane (3). In all cases, the cytoplasmiccomponent IIAB (AB) is in contact with its membrane-located part-ners, but without being directly involved in a receptor function or in animmunity function. CW, cell wall; CM, cytoplasmic membrane. Themodel is based on published work (41, 47, 103) and unpublished work(Diep and Holo, unpublished).
a novel toxin active against eukaryotic and prokaryotic cells. Cell Microbiol.5:661–669.
17. Coburn, P. S., C. M. Pillar, B. D. Jett, W. Haas, and M. S. Gilmore. 2004.Enterococcus faecalis senses target cells and in response expresses cytolysin.Science 306:2270–2272.
18. Cookson, A. L., S. J. Noel, W. J. Kelly, and G. T. Attwood. 2004. The use ofPCR for the identification and characterisation of bacteriocin genes frombacterial strains isolated from rumen or caecal contents of cattle and sheep.FEMS Microbiol. Ecol. 48:1199–1207.
19. Cotter, P. D., C. Hill, and R. P. Ross. 2005. Bacteriocins: developing innateimmunity for food. Nat. Rev. Microbiol. 3:777–788.
20. Criado, R., D. B. Diep, A. Aakra, J. Gutierrez, I. F. Nes, P. E. Hernandez,and L. M. Cintas. 2006. Complete sequence of the enterocin Q-encodingplasmid pCIZ2 from the multiple bacteriocin producer Enterococcus fae-cium L50 and genetic characterization of enterocin Q. Production andimmunity. Appl. Environ. Microbiol. 72:6653–6666.
21. de Jong, A., S. A. F. T. van Hijum, J. J. E. Bijlsma, J. Kok, and O. P.Kuipers. 2006. BAGEL: a web-based bacteriocin genome mining tool.Nucleic Acids Res. 34:273–279.
22. De Kwaadsteniet, M., T. Fraser, C. A. Van Reenen, and L. M. Dicks. 2006.Bacteriocin T8, a novel class IIa sec-dependent bacteriocin produced byEnterococcus faecium T8, isolated from vaginal secretions of children in-fected with human immunodeficiency virus. Appl. Environ. Microbiol. 72:4761–4766.
23. Diep, D. B., L. Godager, D. Brede, and I. F. Nes. 2006. Data mining andcharacterization of a novel pediocin-like bacteriocin system from the ge-nome of Pediococcus pentosaceus ATCC 25745. Microbiology 152:1649–1659.
24. Diep, D. B., L. S. Havarstein, and I. F. Nes. 1995. A bacteriocin-like peptideinduces bacteriocin synthesis in Lactobacillus plantarum C11. Mol. Micro-biol. 8:631–639.
25. Dirix, G., P. Monsieurs, B. Dombrecht, R. Daniels, K. Marchal, J. Vander-leyden, and J. Michiels. 2004. Peptide signal molecules and bacteriocins inGram-negative bacteria: a genome-wide in silico screening for peptidescontaining a double-glycine leader sequence and their cognate transporters.Peptides 25:1425–1440.
26. Donvito, B., J. Etienne, L. Denoroy, T. Greenland, Y. Benito, and F.Vandenesch. 1997. Synergistic hemolytic activity of Staphylococcus lug-dunensis is mediated by three peptides encoded by a non-agr genetic locus.Infect. Immun. 65:95–100.
27. Drider, D., G. Fimland, Y. Hechard, L. M. McMullen, and H. Prevost. 2006.The continuing story of class IIa bacteriocins. Microbiol. Mol. Biol. Rev.70:564–582.
28. Eguchi, T., K. Kaminaka, J. Shima, S. Kawamoto, K. Mori, S. H. Choi, K.Doi, S. Ohmomo, and S. Ogata. 2001. Isolation and characterization ofenterocin SE-K4 produced by thermophilic enterococci, Enterococcus fae-calis K-4. Biosci. Biotechnol. Biochem. 65:247–253.
29. Eijsink, V. G., L. Axelsson, D. B. Diep, L. S. Havarstein, H. Holo, and I. F.Nes. 2002. Production of class II bacteriocins by lactic acid bacteria; anexample of biological warfare and communication. Antonie Leeuwenhoek81:639–654.
30. Eijsink, V. G., M. Skeie, P. H. Middelhoven, M. B. Brurberg, and I. F. Nes.1998. Comparative studies of class IIa bacteriocins of lactic acid bacteria.Appl. Environ. Microbiol. 64:3275–3281.
31. Ennahar, S., T. Sashihara, K. Sonomoto, and A. Ishizaki. 2000. Class IIabacteriocins: biosynthesis, structure and activity. FEMS Microbiol. Rev.24:85–106.
32. Fimland, G., O. R. Blingsmo, K. Sletten, G. Jung, I. F. Nes, and J. Nissen-Meyer. 1996. New biologically active hybrid bacteriocins constructed bycombining regions from various pediocin-like bacteriocins: the C-terminalregion is important for determining specificity. Appl. Environ. Microbiol.62:3313–3318.
33. Foulquie Moreno, M. R., P. Sarantinopoulos, E. Tsakalidou, and L. DeVuyst. 2006. The role and application of enterococci in food and health. Int.J. Food Microbiol. 106:1–24.
34. Franz, C. M., A. Grube, A. Herrmann, H. Abriouel, J. Starke, A. Lombardi,B. Tauscher, and W. H. Holzapfel. 2002. Biochemical and genetic charac-terization of the two-peptide bacteriocin enterocin 1071 produced by En-terococcus faecalis FAIR-E 309. Appl. Environ. Microbiol. 68:2550–2554.
35. Franz, C. M., M. E. Stiles, K. H. Schleifer, and W. H. Holzapfel. 2003.Enterococci in foods—a conundrum for food safety. Int. J. Food Microbiol.88:105–122.
36. Franz, C. M., R. W. Worobo, L. E. Quadri, U. Schillinger, W. H. Holzapfel,J. C. Vederas, and M. E. Stiles. 1999. Atypical genetic locus associated withconstitutive production of enterocin B by Enterococcus faecium BFE 900.Appl. Environ. Microbiol. 65:2170–2178.
37. Fuller, J. D., A. C. Camus, C. L. Duncan, V. Nizet, D. J. Bast, R. L. Thune,D. E. Low, and J. C. De Azavedo. 2002. Identification of a streptolysinS-associated gene cluster and its role in the pathogenesis of Streptococcusiniae disease. Infect. Immunol. 70:5730–5739.
38. Galvez, A., G. Gimenez-Gallego, M. Maqueda, and E. Valdivia. 1989. Pu-rification and amino acid composition of peptide antibiotic AS-48 produced
39. Galvez, A., E. Valdivia, H. Abriouel, E. Camafeita, E. Mendez, M. Mar-tinez-Bueno, and M. Maqueda. 1998. Isolation and characterization ofenterocin EJ97, a bacteriocin produced by Enterococcus faecalis EJ97.Arch. Microbiol. 171:59–65.
40. Georgalaki, M. D., E. Van Den Berghe, D. Kritikos, B. Devreese, J. VanBeeumen, G. Kalantzopoulos, L. De Vuyst, and E. Tsakalidou. 2002. Mace-docin, a food-grade lantibiotic produced by Streptococcus macedonicusACA-DC 198. Appl. Environ. Microbiol. 68:5891–5903.
41. Gravesen, A., M. Ramnath, K. B. Rechinger, N. Andersen, L. Jansch, Y.Hechard, J. W. Hastings, and S. Knochel. 2002. High-level resistance toclass IIa bacteriocins is associated with one general mechanism in Listeriamonocytogenes. Microbiology 148:2361–2369.
42. Gronroos, L., M. Saarela, J. Matto, U. Tanner-Salo, A. Vuorela, and S.Alaluusua. 1998. Mutacin production by Streptococcus mutans may promotetransmission of bacteria from mother to child. Infect. Immun. 66:2595–2600.
43. Guchi, T., K. Kaminaka, J. Shima, S. Kawamoto, K. Mori, S.-H. Choi, K.Doi, S. Ohmomo, and S. Ogata. 2002. Isolation and characterization ofenterocin SE-K4 produced by thermophilic enterococci, Enterococcus fae-calis K-4. Biosci. Biotechnol. Biochem. 65:247–253.
44. Hale, J. D., Y. T. Ting, R. W. Jack, J. R. Tagg, and N. C. Heng. 2005.Bacteriocin (mutacin) production by Streptococcus mutans genome se-quence reference strain UA159: elucidation of the antimicrobial repertoireby genetic dissection. Appl. Environ. Microbiol. 71:7613–7617.
45. Havarstein, L. S., D. B. Diep, and I. F. Nes. 1995. A family of bacteriocinABC transporters carry out proteolytic processing of their substrates con-comitant with export. Mol. Microbiol. 16:229–240.
46. Havarstein, L. S., H. Holo, and I. F. Nes. 1994. The leader peptide of colicinV shares consensus sequences with leader peptides that are common amongpeptide bacteriocins produced by gram-positive bacteria. Microbiology 140:2383–2389.
47. Hechard, Y., and H. G. Sahl. 2002. Mode of action of modified and un-modified bacteriocins from Gram-positive bacteria. Biochimie 84:545–557.
48. Heng, N. C., J. R. Tagg, and G. R. Tompkins. 29 September 2006. Com-petence-dependent bacteriocin production by Streptococcus gordonii DL1(Challis). J. Bacteriol. doi:10.1128/JB.01174–06.
49. Heng, N. C. K., and J. R. Tagg. 2006. What’s in a name? Class distinctionfor bacteriocins. Nat. Rev. Microbiol. doi:10.1038/nrmicro1273-c1.
50. Hillman, J. D., J. Novak, E. Sagura, J. A. Gutierrez, T. A. Brooks, P. J.Crowley, M. Hess, A. Azizi, K. Leung, D. Cvitkovitch, and A. S. Bleiweis.1998. Genetic and biochemical analysis of mutacin 1140, a lantibiotic fromStreptococcus mutans. Infect. Immun. 66:2743–2749.
51. Holo, H., O. Nilssen, and I. F. Nes. 1991. Lactococcin A, a new bacteriocinfrom Lactococcus lactis subsp. cremoris: isolation and characterization ofthe protein and its gene. J. Bacteriol. 173:3879–3887.
52. Hols, P., F. Hancy, L. Fontaine, B. Grossiord, D. Prozzi, N. Leblond-Bourget, B. Decaris, A. Bolotin, C. Delorme, S. Dusko Ehrlich, E. Guedon,V. Monnet, P. Renault, and M. Kleerebezem. 2005. New insights in themolecular biology and physiology of Streptococcus thermophilus revealed bycomparative genomics. FEMS Microbiol. Rev. 29:435–463.
53. Humar, D., V. Datta, D. J. Bast, B. Beall, J. C. S. De Azavedo, and V. Nizet.2002. Streptolysin S and necrotising infections produced by group G strep-tococcus. Lancet 359:124–129.
54. Hyink, O., M. Balakrishnan, and J. R. Tagg. 2005. Streptococcus rattusstrain BHT produces both a class I two-component lantibiotic and a class IIbacteriocin. FEMS Microbiol. Lett. 252:235–241.
55. Hynes, W. L., J. J. Ferretti, and J. R. Tagg. 1993. Cloning of the geneencoding streptococcin A-FF22, a novel lantibiotic produced by Streptococ-cus pyogenes, and determination of its nucleotide sequence. Appl. Environ.Microbiol. 59:1969–1971.
56. Haas, W., and M. S. Gilmore. 1999. Molecular nature of a novel bacterialtoxin: the cytolysin of Enterococcus faecalis. Med. Microbiol. Immunol.187:183–190.
57. Haas, W., B. D. Shepard, and M. S. Gilmore. 2002. Two-component reg-ulator of Enterococcus faecalis cytolysin responds to quorum-sensing auto-induction. Nature 415:84–87.
58. Inoue, T., H. Tomita, and Y. Ike. 2006. Bac 32, a novel bacteriocin widelydisseminated among clinical isolates of Enterococcus faecium. Antimicrob.Agents Chemother. 50:1202–1212.
59. Jack, R. W., G. Bierbaum, and H.-G. Sahl. 1998. Lantibiotics and relatedpeptides. Springer-Verlag, Berlin, Germany.
60. Jack, R. W., A. Carne, J. Metzger, S. Stefanovic, H. G. Sahl, G. Jung, andJ. Tagg. 1994. Elucidation of the structure of SA-FF22, a lanthionine-containing antibacterial peptide produced by Streptococcus pyogenes strainFF22. Eur. J. Biochem. 220:455–462.
61. Jett, B. D., M. M. Huycke, and M. S. Gilmore. 1994. Virulence of entero-cocci. Clin. Microbiol. Rev. 7:462–478.
62. Joosten, H. M., M. Nunez, B. Devreese, J. Van Beeumen, and J. D. Marugg.1996. Purification and characterization of enterocin 4, a bacteriocin pro-
duced by Enterococcus faecalis INIA 4. Appl. Environ. Microbiol. 62:4220–4223.
63. Joosten, H. M., E. Rodriguez, and M. Nunez. 1997. PCR detection ofsequences similar to the AS-48 structural gene in bacteriocin-producingenterococci. Lett. Appl. Microbiol. 24:40–42.
64. Jung, G. 1991. Lantibiotics: a survey, p. 1–35. In G. Jung and H.-G. Sahl(ed.), Nisin and novel lantibiotics. ESCOM Science Publishers, Leiden, TheNetherlands.
65. Jurkovic, D., L. Krizkova, R. Dusinsky, A. Belicova, M. Sojka, J. Krajcovic,and L. Ebringer. 2006. Identification and characterization of enterococcifrom bryndza cheese. Lett. Appl. Microbiol. 42:553–559.
66. Kanatani, K., T. Tahara, M. Oshimura, K. Sano, and C. Umezawa. 1995.Identification of the replication region of Lactobacillus acidophilus plasmidpLA103. FEMS Microbiol. Lett. 133:127–130.
67. Kang, J. H., and M. S. Lee. 2005. Characterization of a bacteriocin pro-duced by Enterococcus faecium GM-1 isolated from an infant. J. Appl.Microbiol. 98:1169–1176.
68. Karaya, K., T. Shimizu, and A. Taketo. 2001. New gene cluster for lantibi-otic streptin possibly involved in streptolysin S formation. J. Biochem.129:769–775.
69. Kawamoto, S., J. Shima, R. Sato, T. Eguchi, S. Ohmomo, J. Shibato, N.Horikoshi, K. Takeshita, and T. Sameshima. 2002. Biochemical and geneticcharacterization of mundticin KS, an antilisterial peptide produced by En-terococcus mundtii NFRI 7393. Appl. Environ. Microbiol. 68:3830–3840.
70. Kayaoglu, G., and D. Orstavik. 2004. Virulence factors of Enterococcusfaecalis: relationship to endodontic disease. Crit. Rev. Oral Biol. Med.15:308–320.
71. Klaenhammer, T. R. 1993. Genetics of bacteriocins produced by lactic acidbacteria. FEMS Microbiol. Rev. 12:39–85.
72. Kreth, J., J. Merritt, W. Shi, and F. Qi. 2005. Co-ordinated bacteriocinproduction and competence development: a possible mechanism for takingup DNA from neighbouring species. Mol. Microbiol. 57:392–404.
73. Krull, R. E., P. Chen, J. Novak, M. Kirk, S. Barnes, J. Baker, N. R. Krishna,and P. W. Caufield. 2000. Biochemical structural analysis of the lantibioticmutacin II. J. Biol. Chem. 275:15845–15850.
74. Laukova, A., S. Czikkova, Z. Vasilkova, P. Juris, and M. Marekova. 1998.Occurrence of bacteriocin production among environmental enterococci.Lett. Appl. Microbiol. 27:178–182.
75. Laukova, A., and M. Marekova. 2001. Production of bacteriocins by differ-ent enterococcal isolates. Folia Microbiol. 46:49–52.
76. Macovei, L., and L. Zurek. 2006. Ecology of antibiotic resistance genes:characterization of enterococci from houseflies collected in food settings.Appl. Environ. Microbiol. 72:4028–4035.
77. Maqueda, M., A. Galvez, M. M. Bueno, M. J. Sanchez-Barrena, C. Gonzalez,A. Albert, M. Rico, and E. Valdivia. 2004. Peptide AS-48: prototype of a newclass of cyclic bacteriocins. Curr. Protein Pept. Sci. 5:399–416.
78. Marciset, O., M. C. Jeronimus-Stratingh, B. Mollet, and B. Poolman. 1997.Thermophilin 13, a nontypical antilisterial poration complex bacteriocin,that functions without a receptor. J. Biol. Chem. 272:14277–14284.
79. Marekova, M., A. Laukova, L. DeVuyst, M. Skaugen, and I. F. Nes. 2003.Partial characterization of bacteriocins produced by environmental strainEnterococcus faecium EK13. J. Appl. Microbiol. 94:523–530.
80. Martin, N. I., T. Sprules, M. R. Carpenter, P. D. Cotter, C. Hill, R. P. Ross,and J. C. Vederas. 2004. Structural characterization of lacticin 3147, atwo-peptide lantibiotic with synergistic activity. Biochemistry 43:3049–3056.
81. Martin-Platero, A. M., E. Valdivia, M. Ruiz-Rodriguez, J. J. Soler, M.Martin-Vivaldi, M. Maqueda, and M. Martinez-Bueno. 2006. Character-ization of antimicrobial substances produced by Enterococcus faecalis MRR10-3, isolated from the uropygial gland of the hoopoe (Upupa epops). Appl.Environ. Microbiol. 72:4245–4249.
82. Martinez-Bueno, M., M. Maqueda, A. Galvez, B. Samyn, J. Van Beeumen,J. Coyette, and E. Valdivia. 1994. Determination of the gene sequence andthe molecular structure of the enterococcal peptide antibiotic AS-48. J.Bacteriol. 176:6334–6339.
83. McAuliffe, O., R. P. Ross, and C. Hill. 2001. Lantibiotics: structure, biosyn-thesis and mode of action. FEMS Microbiol. Rev. 25:285–308.
84. Mindich, L. 1966. Bacteriocins of Diplococcus pneumoniae. I. Antagonisticrelationships and genetic transformations. J. Bacteriol. 92:1090–1098.
85. Moretro, T., K. Naterstad, E. Wang, I. M. Aasen, S. Chaillou, M. Zagorec,and L. Axelsson. 2005. Sakacin P non-producing Lactobacillus sakei strainscontain homologues of the sakacin P gene cluster. Res. Microbiol. 156:949–960.
86. Mota-Meira, M., C. Lacroix, G. LaPointe, and M. C. Lavoie. 1997. Purifi-cation and structure of mutacin B-Ny266: a new lantibiotic produced byStreptococcus mutans. FEBS Lett. 410:275–279.
87. Nes, I. F., and V. G. H. Eijsink. 1999. Regulation of group II peptidebacteriocin synthesis by quorum-Sensing mechansims, p. 175–192. In G. M.Dunny and S. C. Winans (ed.), Cell-cell signaling in bacteria. ASM Press,Washington, DC.
88. Nes, I. F., and H. Holo. 2000. Class II antimicrobial peptides from lacticacid bacteria. Peptide Sci. 55:50–61.
89. Nes, I. F., and O. Johnsborg. 2004. Exploration of antimicrobial potential inLAB by genomics. Curr. Opin. Biotechnol. 15:100–104.
90. Netz, D. J., R. Pohl, A. G. Beck-Sickinger, T. Selmer, A. J. Pierik, C. BastosMdo, and H. G. Sahl. 2002. Biochemical characterisation and genetic anal-ysis of aureocin A53, a new, atypical bacteriocin from Staphylococcus au-reus. J. Mol. Biol. 319:745–756.
91. Netz, D. J., H. G. Sahl, R. Marcelino, J. dos Santos Nascimento, S. S. deOliveira, M. B. Soares, and M. do Carmo de Freire Bastos. 2001. Molecularcharacterisation of aureocin A70, a multi-peptide bacteriocin isolated fromStaphylococcus aureus. J. Mol. Biol. 311:939–949.
92. Nicolas, G., H. Morency, G. LaPointe, and M. C. Lavoie. 2006. MutacinH-29B is identical to mutacin II (J-T8). BMC Microbiol. 6:36.
93. Nilsen, T., I. F. Nes, and H. Holo. 1998. An exported inducer peptideregulates bacteriocin production in Enterococcus faecium CTC492. J. Bac-teriol. 180:1848–1854.
94. Nissen-Meyer, J., H. Holo, L. S. Havarstein, K. Sletten, and I. F. Nes. 1992.A novel lactococcal bacteriocin whose activity depends on the complemen-tary action of two peptides. J. Bacteriol. 174:5686–5692.
95. Nizet, V., B. Beall, D. J. Bast, V. Datta, L. Kilburn, D. E. Low, and J. C. DeAzavedo. 2000. Genetic locus for streptolysin S production by group Astreptococcus. Infect. Immun. 68:4245–4254.
96. O’Keeffe, T., C. Hill, and R. P. Ross. 1999. Characterization and heterolo-gous expression of the genes encoding enterocin A production, immunity,and regulation in Enterococcus faecium DPC1146. Appl. Environ. Micro-biol. 65:1506–1515.
97. Pag, U., and H. G. Sahl. 2002. Multiple activities in lantibiotics—models forthe design of novel antibiotics. Curr. Pharm. Des. 8:815–833.
98. Park, S. H., K. Itoh, and T. Fujisawa. 2003. Characteristics and identifica-tion of enterocins produced by Enterococcus faecium JCM 5804T. J. Appl.Microbiol. 95:294–300.
99. Paulsen, I. T., L. Banerjei, G. S. Myers, K. E. Nelson, R. Seshadri, T. D.Read, D. E. Fouts, J. A. Eisen, S. R. Gill, J. F. Heidelberg, H. Tettelin, R. J.Dodson, L. Umayam, L. Brinkac, M. Beanan, S. Daugherty, R. T. DeBoy, S.Durkin, J. Kolonay, R. Madupu, W. Nelson, J. Vamathevan, B. Tran, J.Upton, T. Hansen, J. Shetty, H. Khouri, T. Utterback, D. Radune, K. A.Ketchum, B. A. Dougherty, and C. M. Fraser. 2003. Role of mobile DNAin the evolution of vancomycin-resistant Enterococcus faecalis. Science 299:2071–2074.
100. Qi, F., P. Chen, and P. W. Caufield. 2001. The group I strain of Strepto-coccus mutans, UA140, produces both the lantibiotic mutacin I and anonlantibiotic bacteriocin, mutacin IV. Appl. Environ. Microbiol. 67:15–21.
101. Qi, F., P. Chen, and P. W. Caufield. 2000. Purification and biochemicalcharacterization of mutacin I from the group I strain of Streptococcusmutans, CH43, and genetic analysis of mutacin I biosynthesis genes. Appl.Environ. Microbiol. 66:3221–3229.
102. Qi, F., P. Chen, and P. W. Caufield. 1999. Purification of mutacin III fromgroup III Streptococcus mutans UA787 and genetic analyses of mutacin IIIbiosynthesis genes. Appl. Environ. Microbiol. 65:3880–3887.
103. Ramnath, M., S. Arous, A. Gravesen, J. W. Hastings, and Y. Hechard. 2004.Expression of mptC of Listeria monocytogenes induces sensitivity to class IIabacteriocins in Lactococcus lactis. Microbiology 150:2663–2668.
104. Rawlinson, E. L., I. F. Nes, and M. Skaugen. 2005. Identification of theDNA-binding site of the Rgg-like regulator LasX within the lactocin Spromoter region. Microbiology 151:813–823.
105. Rawlinson, E. L., I. F. Nes, and M. Skaugen. 2002. LasX, a transcriptionalregulator of the lactocin S biosynthetic genes in Lactobacillus sakei L45, actsboth as an activator and a repressor. Biochimie 84:559–567.
106. Rodriguez, J. M., M. I. Martinez, N. Horn, and H. M. Dodd. 2003. Heter-ologous production of bacteriocins by lactic acid bacteria. Int. J. FoodMicrobiol. 80:101–116.
107. Ross, K. F., C. W. Ronson, and J. R. Tagg. 1993. Isolation and character-ization of the lantibiotic salivaricin A and its structural gene salA fromStreptococcus salivarius 20P3. Appl. Environ. Microbiol. 59:2014–2021.
108. Ross, R. P., M. Galvin, O. McAuliffe, S. M. Morgan, M. P. Ryan, D. P.Twomey, W. J. Meaney, and C. Hill. 1999. Developing applications forlactococcal bacteriocins. Antonie Leeuwenhoek 76:337–346.
109. Ryan, M. P., R. W. Jack, M. Josten, H. G. Sahl, G. Jung, R. P. Ross, andC. Hill. 1999. Extensive post-translational modification, including serine toD-alanine conversion, in the two-component lantibiotic, lacticin 3147.J. Biol. Chem. 274:37544–37550.
110. Ryan, M. P., W. J. Meaney, R. P. Ross, and C. Hill. 1998. Evaluation oflacticin 3147 and a teat seal containing this bacteriocin for inhibition ofmastitis pathogens. Appl. Environ. Microbiol. 64:2287–2290.
111. Saavedra, L., C. Minahk, A. P. de Ruiz Holgado, and F. Sesma. 2004.Enhancement of the enterocin CRL35 activity by a synthetic peptide de-rived from the NH2-terminal sequence. Antimicrob. Agents Chemother.48:2778–2781.
112. Sanchez-Hidalgo, M., M. Maqueda, A. Galvez, H. Abriouel, E. Valdivia,and M. Martinez-Bueno. 2003. The genes coding for enterocin EJ97 pro-duction by Enterococcus faecalis EJ97 are located on a conjugative plasmid.Appl. Environ. Microbiol. 69:1633–1641.
113. Schlegel, R., and H. D. Slade. 1972. Bacteriocin production by transform-able group H streptococci. J. Bacteriol. 112:824–829.
114. Schlegel, R., and H. D. Slade. 1973. Properties of a Streptococcus sanguis(group H) bacteriocin and its separation from the competence factor oftransformation. J. Bacteriol. 115:655–661.
115. Semedo, T., M. Almeida Santos, P. Martins, M. F. Silva Lopes, J. J.Figueiredo Marques, R. Tenreiro, and M. T. Barreto Crespo. 2003. Com-parative study using type strains and clinical and food isolates to examinehemolytic activity and occurrence of the cyl operon in enterococci. J. Clin.Microbiol. 41:2569–2576.
116. Sharma, R. M., and R. A. Packer. 1970. Occurrence and ecologic featuresof Streptococcus uberis in the dairy cow. Am. J. Vet. Res. 31:1197–1202.
117. Shen, K., J. Gladitz, P. Antalis, B. Dice, B. Janto, R. Keefe, J. Hayes, A.Ahmed, R. Dopico, N. Ehrlich, J. Jocz, L. Kropp, S. Yu, L. Nistico, D. P.Greenberg, K. Barbadora, R. A. Preston, J. C. Post, G. D. Ehrlich, and F. Z.Hu. 2006. Characterization, distribution, and expression of novel genesamong eight clinical isolates of Streptococcus pneumoniae. Infect. Immun.74:321–330.
118. Skaugen, M., J. Nissen-Meyer, G. Jung, S. Stevanovic, K. Sletten, C. Inger,M. Abildgaard, and I. F. Nes. 1994. In vivo conversion of L-serine toD-alanine in a ribosomally synthesized polypeptide. J. Biol. Chem. 269:27183–27185.
119. Tagg, J. R. 2004. Prevention of streptococcal pharyngitis by anti-Strepto-coccus pyogenes bacteriocin-like inhibitory substances (BLIS) produced byStreptococcus salivarius. Indian J. Med. Res. 119:13–16.
120. Tettelin, H., K. E. Nelson, I. T. Paulsen, J. A. Eisen, T. D. Read, S. Peterson,J. Heidelberg, R. T. DeBoy, D. H. Haft, R. J. Dodson, A. S. Durkin, M.Gwinn, J. F. Kolonay, W. C. Nelson, J. D. Peterson, L. A. Umayam, O.White, S. L. Salzberg, M. R. Lewis, D. Radune, E. Holtzapple, H. Khouri,A. M. Wolf, T. R. Utterback, C. L. Hansen, L. A. McDonald, T. V.Feldblyum, S. Angiuoli, T. Dickinson, E. K. Hickey, I. E. Holt, B. J. Loftus,F. Yang, H. O. Smith, J. C. Venter, B. A. Dougherty, D. A. Morrison, S. K.Hollingshead, and C. M. Fraser. 2001. Complete genome sequence of avirulent isolate of Streptococcus pneumoniae. Science 293:498–506.
121. Tomita, H., S. Fujimoto, K. Tanimoto, and Y. Ike. 1997. Cloning andgenetic and sequence analyses of the bacteriocin 21 determinant encodedon the Enterococcus faecalis pheromone-responsive conjugative plasmidpPD1. J. Bacteriol. 179:7843–7855.
122. Tomita, H., S. Fujimoto, K. Tanimoto, and Y. Ike. 1996. Cloning andgenetic organization of the bacteriocin 31 determinant encoded on theEnterococcus faecalis pheromone-responsive conjugative plasmid pYI17. J.Bacteriol. 178:3585–3593.
123. Turner, D. L., L. Brennan, H. E. Meyer, C. Lohaus, C. Siethoff, H. S. Costa,B. Gonzalez, H. Santos, and J. E. Suarez. 1999. Solution structure ofplantaricin C, a novel lantibiotic. Eur. J. Biochem. 264:833–839.
124. van der Ploeg, J. R. 2005. Regulation of bacteriocin production in Strepto-coccus mutans by the quorum-sensing system required for development ofgenetic competence. J. Bacteriol. 187:3980–3989.
125. Ward, D. J., and G. A. Somkuti. 1995. Characterization of a bacteriocin
produced by Streptococcus thermophilus ST134. Appl. Microbiol. Biotech-nol. 43:330–335.
126. Watson, D. C., M. Yaguchi, J. G. Bisaillon, R. Beaudet, and R. Morosoli.1988. The amino acid sequence of a gonococcal growth inhibitor fromStaphylococcus haemolyticus. Biochem. J. 252:87–93.
127. Wescombe, P. A., and J. R. Tagg. 2003. Purification and characterization ofstreptin, a type A1 lantibiotic produced by Streptococcus pyogenes. Appl.Environ. Microbiol. 69:2737–2747.
128. Wescombe, P. A., M. Upton, K. P. Dierksen, N. L. Ragland, S. Sivabalan,R. E. Wirawan, M. A. Inglis, C. J. Moore, G. V. Walker, C. N. Chilcott, H. F.Jenkinson, and J. R. Tagg. 2006. Production of the lantibiotic salivaricin Aand its variants by oral streptococci and use of a specific induction assay todetect their presence in human saliva. Appl. Environ. Microbiol. 72:1459–1466.
129. Whitford, M. F., M. A. McPherson, R. J. Forster, and R. M. Teather. 2001.Identification of bacteriocin-like inhibitors from rumen Streptococcus spp.and isolation and characterization of bovicin 255. Appl. Environ. Microbiol.67:569–574.
130. Wiedemann, I., T. Bottiger, R. R. Bonelli, A. Wiese, S. O. Hagge, T.Gutsmann, U. Seydel, L. Deegan, C. Hill, P. Ross, and H. G. Sahl. 2006.The mode of action of the lantibiotic lacticin 3147—a complex mechanisminvolving specific interaction of two peptides and the cell wall precursorlipid II. Mol. Microbiol. 61:285–296.
131. Wirawan, R. E., R. W. Jack, and J. R. Tagg. 2006. The antimicrobial(bacteriocin) repertoire of Streptococcus uberis, abstr. B14. StreptococcalGenet. Conf., Saint-Malo, France, 18 to 21 June 2006. American Society forMicrobiology, Washington, DC.
132. Wirawan, R. E., N. A. Klesse, R. W. Jack, and J. R. Tagg. 2006. Molecularand genetic characterization of a novel nisin variant produced by Strepto-coccus uberis. Appl. Environ. Microbiol. 72:1148–1156.
133. Xiao, H., X. Chen, M. Chen, S. Tang, X. Zhao, and L. Huan. 2004. BovicinHJ50, a novel lantibiotic produced by Streptococcus bovis HJ50. Microbi-ology 150:103–108.
134. Yamamoto, Y., Y. Togawa, M. Shimosaka, and M. Okazaki. 2003. Purifi-cation and characterization of a novel bacteriocin produced by Enterococ-cus faecalis strain RJ-11. Appl. Environ. Microbiol. 69:5746–5753.
135. Yonezawa, H., and H. K. Kuramitsu. 2005. Genetic analysis of a uniquebacteriocin, Smb, produced by Streptococcus mutans GS5. Antimicrob.Agents Chemother. 49:541–548.
136. Zadoks, R. N., H. G. Allore, H. W. Barkema, O. C. Sampimon, Y. T. Grohn,and Y. H. Schukken. 2001. Analysis of an outbreak of Streptococcus uberismastitis. J. Dairy Sci. 84:590–599.
137. Zadoks, R. N., L. L. Tikofsky, and K. J. Boor. 2005. Ribotyping of Strep-tococcus uberis from a dairy’s environment, bovine feces and milk. Vet.Microbiol. 109:257–265.
138. Zendo, T., N. Eungruttanagorn, S. Fujioka, Y. Tashiro, K. Nomura, Y.Sera, G. Kobayashi, J. Nakayama, A. Ishizaki, and K. Sonomoto. 2005.Identification and production of a bacteriocin from Enterococcus mundtiiQU 2 isolated from soybean. J. Appl. Microbiol. 99:1181–1190.
