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Protein J
DOI 10.1007/s10930-010-9270-4

A New Structure-based Classification of Gram-positive
Bacteriocins
Abdelmajid Zouhir • Riadh Hammami
Ismail Fliss • Jeannette Ben Hamida



Springer Science+Business Media, LLC 2010

Abstract Bacteriocins are ribosomally-synthesized peptides or proteins produced by a wide range of bacteria. The
antimicrobial activity of this group of natural substances
against foodborne pathogenic and spoilage bacteria has
raised considerable interest for their application in food
preservation. Classifying these bacteriocins in well defined
classes according to their biochemical properties is a major
step towards characterizing these anti-infective peptides
and understanding their mode of action. Actually, the
chosen criteria for bacteriocins’ classification lack consistency and coherence. So, various classification schemes of
bacteriocins resulted various levels of contradiction and
sorting inefficiencies leading to bacteriocins belonging to
more than one class at the same time and to a general lack
of classification of many bacteriocins. Establishing a
coherent and adequate classification scheme for these
bacteriocins is sought after by several researchers in the
field. It is not straightforward to formulate an efficient
classification scheme that encompasses all of the existing
bacteriocins. In the light of the structural data, here we
revisit the previously proposed contradictory classification
and we define new structure-based sequence fingerprints
that support a subdivision of the bacteriocins into 12
groups. The paper lays down a resourceful and consistent
classification approach that resulted in classifying more

A. Zouhir (&) R. Hammami J. B. Hamida
Unite´ de Prote´omie Fonctionnelle et Biopre´servation
Alimentaire, ISSBAT, El Manar, BP 94,
Tunis cedex, 1068 Tunis, Tunisia
e-mail: azouheirb10@yahoo.fr
R. Hammami I. Fliss
Dairy Research Center STELA, Nutraceuticals and Functional
Foods Institute (INAF), Universite´ Laval, Que´bec,
PQ G1K 7P4, Canada

than 70% of bacteriocins known to date and with potential
to identify distinct classes for the remaining unclassified
bacteriocins. Identified groups are characterized by the
presence of highly conserved short amino acid motifs.
Furthermore, unclassified bacteriocins are expected to form
an identified group when there will be sufficient sequences.
Keywords Bacteriocins Classification Sequence
analysis Phylogeny
Abbreviations
DNA
HMMs
MEME
NJ
PDB
RCSB
SciDBMaker

Desoxyribonucleic acid
Hidden Markov Models
Multiple EM for Motif Elicitation
Neighbor-Joining method
Protein Data Bank
Research Structural Bioinformatics
Scientific DataBase Maker

1 Introduction
Bacteriocins are very diverse group of antimicrobial peptides produced by a wide range of bacteria and known for
their inhibitory activity against various human and animal
pathogens. The antimicrobial activity of this group of
natural substances against foodborne pathogenic and
spoilage bacteria has raised considerable interest for their
application in food preservation. The bacteriocins produced
by Gram-positive bacteria have been grouped into different
classes on the basis of some different criteria [12, 17, 19,
22, 28]. Classification of bacteriocins has traditionally been
based either on their activity [8, 19], action of mode [7],

123

A. Zouhir et al.

method of excretion [17], or their lantibiotic amino acid
structure [5]. However, the majority of bacteriocin classes
are identified by different criterion of classification. As
consequence, the current criteria chosen for bacteriocins’
classification lack consistency and coherence leading to
cases where the same bacteriocin is found in more than one
class depending on the author and the criteria adopted for
classification. For instance, some researchers have previously announced that carnobacteriocin BM1 is identical to
piscicocin V1b, piscicolin 126 is identical to piscicocin
V1a [1], and curvacin A is identical to sakacin A [1].
During this study, we found that Leucocin A is identical to
leucocin B Ta11a. According to [20], sakacin P is identical
to bavaricin A and divercin V41 is identical to bavaricin
MN [18], but after verification we found differences
between these molecules.
According to the actual classification, among 107 bacteriocins 40 molecules are classified in one class or subclass, and 20 bacteriocins are set in more than one class or
subclass at the same time. The current established bases of
Gram-positive bacteriocins classifications are fundamentally different. To this extent, clash of opinions was clearly
evident between [17, 19, 23, 31]. While [17, 19, 24]
grouped Gram-positive bacteriocins in four classes, [23]
suggested only three classes of Gram-positive bacteriocins.
It is noteworthy that [23] excluded the fourth class of
bacteriocins proposed by [19] because the compounds of
this class have not been purified. These classification discrepancies are due to the fact that established parameters
for classifying bacteriocins are particularly ambiguous. For
example, The distribution of class I in two subclasses (type
A and type B) based on their different modes of action is
somewhat arbitrary due to the fact that some bacteriocins
with multiple modes of actions can fall under type A or
type B subclass [7]. The sub-classification of class II led to
a great divergence between researchers. Although [17] did
not propose subclasses, class II is divided into IIa and IIb
according to [7], into IIa, IIb and IIc according to [19, 22,
24], and into IIa, IIb, IIc, IId according to [21]. In 2000,
[31] proposed six subclasses: IIa, IIb, IIc, IId, IIe and IIf for
class II. The current criteria chosen for sub-classification of
the bacteriocins of class II seem incoherent, inadequate and
unclear [7]. Given that current criteria of classification are
ambiguous in nature, a great number of new bacteriocins
are not adequately classified, as they can be placed in more
than one sub-class at the same time. One of the main
characteristics of class IIa bacteriocins is their N-terminal
consensus sequence YGNGVXC [21]. Also known as
Pediocin-like [17], class IIa bacteriocins are often defined
as Listeria-active peptides [19]. However, bacteriocins of
different classes including the nisin A/Z (class I) [6],
variacin (class I) [26], lactocin 705 (class IIb) [32] and
subtilosin A (not classified) [33] are active against Listeria

123

spp. [10, 31], yet they don’t contain the motif YGNGVXC.
Authors used different N-terminal consensus sequence to
characterize the sub-class IIa bacteriocins [13, 22]. While
[12] used consensus sequence YGNGVXCX4CXV, [11]
used sequence YGNGVXC, [22] used sequence YYGNGV
XCXKXXCXV(D/N)W(G/A) and [4] used sequence YGN
GV(X)CX4CXVX4A. The enterocin P and bacteriocin 31
contain the conserved YGNGVXC motif [29] and were
characterized as Class IIa according to the classification of
[19, 23]. In contrast, [17] classified these bacteriocins in
Class IIb based on their method of excretion from the cell.
The current study revealed that enterocin SE K4, acidocin, mundticin KS and prebacteriocin 423 contain the
N-terminal consensus sequence characteristic of the class IIa
albeit they were not classified in this sub-class. Conversely,
acidocin A, acidocin 8912, curvacin FS47 and enterocin I
were characterized as class IIa bacteriocins [7] although they
do not contain the consensus motif characteristic of this
group. While [10] set leucocin A, sakacin P, sakacin A,
curvacin A, carnobacteriocin BM1, bacteriocin 31, enterocin P and listeriocin 7A in class IIa, [31] classified them in
class IIb. Moreover, bacteriocin 31 and enterocin P were
classified in bacteriocins of sec-dependent according to [7].
Other examples of classification inconsistency include the
lactococcin G beta, PlnJ and lactacin F lafX that are
simultaneously placed in class IIb [22] and class IIe [31].
Table 1 shows more examples of bacteriocins that can be
placed into more than one subgroup.
This lack of coherence and consistency in actual classifications motivated us to look for a more insightful
approach based on the structural properties of bacteriocins.
Until now, different classification criteria were used
including function, action of mode and target organisms,
factors that depends on the sequence structure of bacteriocins. In this paper, (1) we present actual classification
methods and their limitations, (2) we analyze structural
similarities between Gram-positive bacteriocins and evaluate their phylogenetic evolution and finally, (3) we identify distinct groups and subgroups that are individually
characterized by consensus sequence motif.

2 Materials and Methods
Structural parameters were extracted from the BACTIBASE database [15] and analyzed using the software
SciDBMaker, a tool for protein sequences analysis [16].
Multiple sequence alignments of 107 Gram-positive bacteriocins was made using the CLUSTALW program [30]
and further refined manually. The parameters used in
CLUSTALW program were as follows: Gap opening, 10;
gap extension, 0.2; delay divergent sequence, 30%; DNA
transition weight, 0.5; protein weight matrix, and Gonnet

Classification of Gram-positive Bacteriocins
Table 1 Bacteriocins placed in more than one subclass according the
current classification
Bacteriocin

Current class

Ref.

Bacteriocin 31

Class IIa

[17]

Carnobacteriocin A

Class IIb

[10]

Class IIc

[23]

IIc Sec-dependent
bacteriocins

[12]

Class IIc

[10]

Class IId

[11]

Non subgrouped bacteriocins [12]
Carnobacteriocin BM1
Curvacin A
Divergicin A
Enterocin B

Class IIa

[17]

Class IIb

[10]

Class IIa

[17]

Class IIb

[10]

Class Iic

[10]

Sec-dependent bacteriocins

[12]

Class IIc

[10]

Class IId

[11]

Non subgrouped bacteriocins [12]
Enterocin P

Lacticin F lafA
Lactacin F lafX
Lactobin A (AmylovorinL4)

Class IIa

[17]

Sec-dependent bacteriocins
Class IIb

[12]
[10]

Class IIc

[11,
23]

Class IIb

[11]

Class IIe

[10]

Class IIb

[18]

Class IIe

[10]

Class IIb

[11]

Class IId

[23]

Non subgrouped bacteriocins [12]
Lactocin 705

Class IIb

[12]

Lactococcin B

Class IId
Class IIc

[23]
[10]

Class IId

[11]

Non-subgrouped bacteriocins [12]
Lactococcin G beta
Leucocin A
Listeriocin 743A
PlnF
PlnJ
Sakacin A
Sakacin P

Class IIb

[18]

Class IIe

[10]

Class IIa

[17]

Class IIb

[10]

class IIa

[17]

Sec-dependent bacteriocins

[12]

Class IIb

[11]

Class IIe

[10]

Class IIb

[18]

Class IIe

[10]

Class IIa

[11]

Class IIb

[10]

Class IIa
Class IIb

[17]
[10]

series. Alignment was manually refined using the GENEDOC (http://www.nrbsc.org/gfx/genedoc/index.html) in
shaded mode. The refinement was conducted based on
similarities of the following groups of amino acids: [T,S—
small nucleophile amino acids], [K, R, H—basic amino
acids], [D, E, N, Q—acidic amino acids and relative
amides], and [L, I, V, M, A, G, P, F, Y, W—hydrophobic
amino acids]. The non-redundant alignment was performed
manually with GENEDOC while considering similarities
between aforementioned groups of amino acids. Based on
the initial alignment a resample was performed by the
generation of 1000 bootstrapped data set using the SEQBOOT of Felsentein’s PHYLIP 3.57c phylogeny inference
package program [14]. Sequence distances of the alignments were calculated using the Dayhoff PAM matrix with
the PROTDIST program [14]. Subsequently, the trees were
constructed by successive clustering of lineages using
the neighbor-joining algorithm as implemented in the
NEIGHBOR program [14]. Their strict consensus tree
was obtained using the CONSENSE program [14]. The
unrooted tree diagram was generated with the FigTree program (http://tree.bio.ed.ac.uk/software/figtree/). All PDB
files were downloaded from the RCSB Protein Data Bank
(http://www.rcsb.org/pdb) and edited with the molecule
analysis and molecule display (PyMOL) program (http://
www.pymol.org). The identification of consensus motifs
was done using MEME program v3.5.4 [27]. Identified
motifs were checked so that incorrect or insignificant matches were discarded. Finally, curated seed alignment containing a set of representative members was used to build a
hidden Markov models (HMMs) profile for each identified
family. A web interface was developed at BACTIBASE
database (http://bactibase.pfba-lab-tun.org/toolsui.php?do=
hmm) that uses HMMER program for profile-HMM searches [9].

3 Results and Discussion
To characterize the molecular evolution of Gram-positive
bacteriocins, we aligned the retrieved amino acid sequences from the BACTIBASE database [15]. A total number
of 107 bacteriocins were analyzed belonging to 14 families,
17 genus and 43 species, as shown in Fig. 1. The structural
alignment of these Gram-positive bacteriocins imposes
the presence of several gaps including gaps situated at the
N-terminal section (residues in position 1 to 13) in the
majority of molecules, gaps at positions 48–53 for almost
all of the second half of peptides, gaps around the residues
40 and 44 in most of the bacteriocins, and gaps at the
C-terminal region from the residues 55 to 75. Other small
gaps of one or some positions are not indicated here.
Approximately, 70% of Gram positive bacteriocins are

123

A. Zouhir et al.

Bacteriocin
Actargadine
Mersacidin
Lacticin 3147A1

10
20
30
40
50
60
70
.........|.........|.........|.........|.........|.........|.........|....
---------------SSGWVCTLTIECGT------V--------ICAC----------------------------------CTFTLPGGGGVCTLTSEC-----------------IC-----------------------------CSTNTFSLSDYWGNNGAWCTLTHEC-M------A--------WCK---------------------------

-1
-1
0

Group 1

Lac
La
La
Sta
Ko
St
St
Ru
St
St

Plantaricin Wa
Bacteriocin J46
Lacticin 481
Nukacin ISK1
Variacin
Streptococcin AFF22
Streptococcin AM49
Ruminococcin A
Mutacin H29B
Mutacin 2

---KCKWWNISCDLGNNGHVCTLSHEC-Q------V--------SCN---------------------------------------KGGSGVIHTISHEVIYNSWNFVF--------TCCS--------------------------------------KGGSGVIHTISHECNMNSWQFVF--------TCCS--------------------------------------KKKSGVIPTVSHDCHMNSFQFVF--------TCCS----------------------------------------GSGVIPTISHECHMNSFQFVF--------TCCS---------------------------------------GKNGVFKTISHECHLNTWAFLA--------TCCS---------------------------------------GKNGVFKTISHECHLNTWAFLA--------TCCS---------------------------------------G-NGVLKTISHECNMNTWQFLF--------TCC--------------------------------------NRWWQGVVPTVSYECRMNSWQHVF--------F----------------------------------------NRWWQGVVPTVSYECRMNSWQHVF--------TCC---------------------------

+2
+2
+2
+4
+1
+3
+3
+1
+2
+2

Group 2

Stm
Stm
Stm

Ancovenin
Duramycin C
Cinnamycin

---------------------------------CVQSCS-----FGPLTWSCDGNTK-------------------------------------------------CANSCS-----YGPLTWSCDGNTK-------------------------------------------------CRQSCS-----FGPFTFVCDGNTK-----------------

0
0
0

Group 3

En
Leu
Li
Lac
En
En
Lac
Leu
Leu
Leu
Lac
Lac
Car
Bs
Car
En
En
Pe
Car
Car
Lac
En
La
En
Car
Car
Lac
Lac
Car

Bacteriocin 31
Leucocin C
Listeriocin 743A
Sakacin P
Mundticin
Mundticin KS
Prebacteriocin 423
Leucocin A
Leucocin BTa11a
Mesentericin Y105
Plantaricin C19
Bavaricin MN
Divercin V41
Coagulin A
Divergicin M35
Enterocin A
Enterocin CRL35
Pediocin PA1
Pisciocin V1a
Piscicolin 126
Bavaricin A
Enterocin SEK4
Lactococcin MMFII
Enterocin P
Carnobacteriocin BM1
Pisciocin V1b
Curvacin A
Sakacin A
Carnobacteriocin B2

------------ATYYGNGLYCNKQKCWVDWNKASREIG----KIIVNG-NVQHGPWAP--R------------------------KNYGNGVHCTKKGCSVDWGYAWTNIANNSVMNGLTG---GNAGWHN---------------------------KSYGNGVQCNKKKCWVDWGSAISTIGNNSAANWATG---GAAGWKS---------------------------KYYGNGVHCGKHSCTVDWGTAIGNIGNNAAANWATG---GNAGWNK---------------------------KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAANLATG---GAAGWSK---------------------------KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAANLATG---GAAGWKS---------------------------KYYGNGVTCGKHSCSVNWGQAFS----CSVSHLANF---GHGKC-----------------------------KYYGNGVHCTKSGCSVNWGEAFS----AGVHRLANG---GNGFW-----------------------------KYYGNGVHCTKSGCSVNWGEAFS----AGVHRLANG---GNGFW-----------------------------KYYGNGVHCTKSGCSVNWGEAAS----AGIHRLANG---GNGFW-----------------------------KYYGNGLSCSKKGCTVNWGQAFS----CGVNRVATA---GHGK-----------------------------TKYYGNGVYCNSKKCWVDWGQAAGGIG----QTVVXG--WLGGAIPG--K-----------------------TKYYGNGVYCNSKKCWVDWGQASGCIG----QTVVGG--WLGGAIPG--KC-----------------------KYYGNGVTCGKHSCSVDWGKATTCIINNGAMAWATG---GHQGTHKC-------------------------TKYYGNGVYCNSKKCWVDWGTAQGCI------DVVIG--QLGGGIPGKGKC------------------TTHSGKYYGNGVYCTKNKCTVDWAKATTCIA----GMSIGG--FLGGAIPG--KC-----------------------KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAANLATG---GAAGWKS---------------------------KYYGNGVTCGKHSCSVDWGKATTCIINNGAMAWATG---GHQGNHKC--------------------------KYYGNGVSCNKNGCTVDWSKAIGIIGNNAAANLTTG---GAAGWNKG--------------------------KYYGNGVSCNKNGCTVDWSKAIGIIGNNAAANLTTG---GAAGWNKG--------------------------KYYGNGVHXGKHSXTVDWGTAIGNIGNNAAANXATG---XNAGG----------------------------ATYYGNGVYCNKQKCWVDWSRARSEIIDRGVKAYVNGFTKVLGGIGG--R------------------------TSYGNGVHCNKSKCWIDVSELETYKA----GTVSN-----PKDILW-------------------------ATRSYGNGVYCNNSKCWVNWGEAKENIA----GIVISG---WASGLAGMGH-----------------------AISYGNGVYCNKEKCWVNKAENKQAIT----GIVIGG---WASSLAGMGH-----------------------AISYGNGVYCNKEKCWVNKAENKQAIT----GIVIGG---WASSLAGMGH-----------------------ARSYGNGVYCNNKKCWVNRGEATQSII----GGMISG---WASGLAGM-------------------------ARSYGNGVYCNNKKCWVNRGEATQSII----GGMISG---WASGLAGM--------------------------VNYGNGVSCSKTKCSVNWGQAFQERYTAGINSFVSG---VASGAGSIGRRP----------

+2
+4
+4
+4
+4
+4
+6
+4
+4
+4
+6
+3
+3
+6
+3
+3
+4
+6
+3
+3
+3
+5
+1
+2
+3
+3
+3
+3
+4

Group 4

Sta
Sta
St
St
St
La
La
Bs

Epidermin
Gallidermin
Mutacin 1140
Mutacin BNy266
Streptin
Nisin A
Nisin Z
Subtilin

---------------IASKFICTP-GCAK-TGSFN---------SYCC----------------------------------------IASKFLCTP-GCAK-TGSFN---------SYCC----------------------------------------FKSWSLCTP-GCAR-TGSFN---------SYCC----------------------------------------FKSWSFCTP-GCAK-TGSFN---------SYCC-----------------------------------------GSRYLCTPGSCWKLVCFTTT---------VK-----------------------------------------ITSISLCTP-GCK--TGALMGCNM----KTATCH---CSIHVSK-----------------------------ITSISLCTP-GCK--TGALMGCNM----KTATCN---CSIHVSK-----------------------------WKSESLCTP-GCV--TGALQTCFL----QTLTCN---C--KISK---------------

+2
+2
+2
+2
+3
+2
+4
+2

Group 5

Sta
Sta

Epicidin 280
Pep 5

--------------SLGPAIKATRQVCPKATRFVTV--SCK-KSDCQ----------------------------------------------KT-----LKATRLFTV--SCKGKNGCK---------------------------

+5
+8

Group 6

Bs
La
Lac

Cytolysin
Lacticin 3147A2
Plantaricin Wbeta

----------------GDVHAQTTWPCATVGVSVAL---CP-TTKCTSQC-------------------------------------TTPATPAISILSAYISTNTCPT-------TKCTRAC---------------------------------------SGIPCTIG---AAVAASIAV---CP-TTKCSKRCGKRKK-------------------

+1
+2
+7

Group 7

Lac
Lac

Acidocin J1132 beta
Plantaricin 1.25 beta

--------------GNPKVAHCASQI----GRST------A-WGAVSGA---------------------------KKKKKKVACTWGNAATAAASGAVXGILGGPTGALAGAI-WGVSQCASNNLHGMH-----------------

+3
+8

Group 8

Car
En

Carnobacteriocin A
Enterocin B

---DQMSDGVNYGKGSSLSKGGAKCGLGIVGGLATIPSGPLGWLAGAAGVINSCMK-----------------ENDHRMPNELN--RPNNLSKGGAKCGAAIAGGLFGIPKGPLAWAAGLANVYSKCN-------------------

+2
+4

Group 9

Lac
Lac
Lac

Gassericin T
Lacticin FlafA
Plantaricin S alfa

--------RNNWAANIGGVGGATVAGWALGNAVCGPACGFVGAHYVPIAWAGVTAATGGFGKIRK----------------RNNWQTNVGGAVGSAMIGATVGGTICGPACAVAGAHYLPILWTAVTAATGGFGKIRK----------------RNKLAYNMGHYAG--------KATIFG-----------LAAWALLA--------------------

+5
+5
+4

Group 10

Lac
Lac
Lac

Acidocin LF221B
Lacticin FlafX
LactobinA

---NKWGNAVIGAATGATRGVSWCRGFG-PWGMTACALG----GAAIGGYLGYKSN--------------------NRWGDTVLSAASGAGTGIKACKSFG-PWGMAICGVG----GAAIGGYFGYTHN--------------------NRWTNAYSAALGCAVPGVKYGKKLGGVWGAVIGGVG----GAAVCGLAGYVRKG-----------------

+6
+3
+6

Group 11

Lac
Car

Lactocin 705
Divergicin 750

-------------GMSGYIQGIPDFLKGYLHGISAAN-------KHKKGRL-----------------------------------KGILGKLGVVQAGVDFVSGVWAG---------IKQSAKDHPNA------------------

+6
+3

Group

Genus
Ac
Bs
La

Fig. 1 Multiple sequence alignment of Gram-positive bacteriocins.
All bacteriocins are taken from the BACTIBASE database. Abbreviated names of producer organism are shown in distinct colors, each
color corresponding to a different genus: Ac Actinoplanes, Bs Bacillus,
La Lactococcus, Lac Lactobacillus, Mi Micrococcus, Ru Ruminococcus, St Streptococcus, Stm Streptomyces, Car Carnobacterium, En

123

Enterococcus, Leu Leuconostoc, Li Listeria, Pe Pediococcus, Sta
Staphylococcus, Cl Clostridium, Br Brochothrix, Pr Propionibacterium. Basic residues are in blue, acidic residues in red, hydrophobic
residues are in green and cysteines are in yellow. The net charge of the
peptide is indicated at the end of each sequence

Classification of Gram-positive Bacteriocins

Group 12

Bs
Lac
La

Sublancin 168
PlnJ
Lactococcin G beta

--------------GLGKAQCAALWLQCASGGTIGCGGG----AVACQNYRQFCR---------------------------------GAWKNFWSSLRKGFYDGEAGR--------AIRR--------------------------------------KKWGWLAWVDP-AYEFIKGFGKG--------AIKEGNKDKWKNI-----------------

+3
+4
+4

Lac
Lac
En
Cl
Lac
Bs
Br
Lac
St
Car
Bs
Bs
Lac
Pr
Lac
Lac
La
St
Lac
Bs
Lac
Lac
Bs
Lac
La
Lac
Lac
Lac
Lac
Lac
En
Leu
Sta
En

Plantaricin NC8a
Plantaricin NC8b
Enterocin Q
Boticin B
Lactocin S
Thuricins
Brochocin C
Curvaticin FS47
Thermohilin A
Divergicin A
Lichenin
Subpeptin JM4B
Curvalicin 28c
Propionicin T1
Curvalicin 28b
Plantaricin Sb
Lactococcin B
Thermohilin 13
Plantaricin A
Subtilosin A
Gassericin A
Curvalicin 28a
Subtilosin
Acidocin B
Lactococcin A
PlnF
PlnK
PlnE
Acidocin A
Acidocin 8912
Enterocin 1071A
Leucocin B
Epilancin K7
Enterocin I

------MDKFEKISTSNLEKISGGDLTTKLWSSWGYYLG----KKARWNLKHPYVQF----------------------MNNLNKFSTLGKSSLSQIEGGSVPTSVYTLGIKILWSAYKHRKTIEKSFNKGFYH----------------------MNFLKNGIAKWMTGAELQAYKKKYGCLP----WEKISC----------------------------------MQKPEIISADLGLCAVNEFVALAAIPGGAATFAVCQMPNLDEIVSNAAYV--------------------------STPVLASVAVSMELLPTASVLYSDVAGCF-KYSAKHHC-----------------------------------------------DWTXWSXLVX---AAC----SVELL------------------------------YSSKDCLKDIG-KGIGAGTVAGAAGGGLAAGLGAI-PGAFVGAHFGVIGG-----------------------YTAKQCLQAIGSCGI-AGTGAGAAGG--PA-------GAFVGAXVVXI---------------------------GALWGAPAGGVGALPGAFVGAHVGAIAGG-FAC-MGGMIGNKFN--------------------------AAPKITQKQKNCVNGQLGGMLAGALGGPGGVVLGGI-GGAIAGGCFN---------------------------------------------------ISLEICXI--------------FHDN-----------------------------------------------XXKEIXHI--------------FHDN----------------------------------------------------------NIPQLT-----P-----------TP----------------VPGGCTYTRSNRDVIGTCKTGSGQFRIRLDCNNAPDKTSVWAKPKVMVSVHCLVGQPRSISFETK
-------------------------VAPFPEQFLX-------------------------------------------------KKKKQSWYAAAGDAIVSFGEGFLNAW--------------------------------------------SLQYVMSAGPYTWYKDTRTGKTICKQTIDTASYTFGVMAEGWGKTFH--------------------------QINWGSVVGHCIGGAIIGGAFSGGAAAG------VGCLVGSGKAIINGL--------------------------AYSLQMGATAIKQVKKLFKKWGW------------------------------------------------------NKGCATCSIGAACLVDGPIPDFEI------AGATGLFGLWG----------------------IYWIADQFGIHLATGTARKLLDAMASGASLGTAFAAILGVTLPAWALAAAGALGATAA------------------------TPVVNPPFLQQT------------------------------------------------------------MKLPVQQVYSVYGGKDLPKGHSHSTMPFLSKLQFLTKIYLLDIHTQPFFI--------------MDKKTKILFEVLYIICIIGPQFILFVTAKNNMYQLVGSFVGIVWFSYIFWYIFFKQHKKM-----------------KLTFIQSTAAGDLYYNTNTHKYVYQQTQNAFG-AAANTIVNGWMGGAAGGFGLHH---------------------VFHAYSARGVRNNYKSAVGPADWVISAVR-----GFIHG---------------------------------------RRSRKNGIGYAIGYAFGAVER--------AVLGGSRDYNK---------------------------------FNRGGYNFGKSVRHVVDAIGS-------VAGIRGILKSIR----------------------KTYYGTNGVHCTKKSLWGKVRLKNVIPGTLCRKQSLPIKQDLKILLGWATGAFGKTFH----------------------------KTHYPTNAWKSLWKGFWES-------LRYTDGF-----------------------MKQYKVLNEKEMKKPIGGESVFSKIGNAVGPAAYWILKGLGNMSDVNQADRINRKKH-----------------------------KGKGFWSWASKATSWLTGPQQPG-------SPLLKKHR---------------------------------SASVLKTSIKVSKKYCKGVTLTCGC--------NITGGK-------------------------------------MGAIAKLVAKFGWPIVKKYYKQIMQFIGEGWAINKIIEWIKKHI----------------

+5
+8
+4
-3
+2
-2
+2
+1
+2
+3
-1
+1
0
+7
-1
+2
+3
+2
+5
-2
+1
0
+6
+6
+4
+5
+5
+6
+12
+3
+7
+4
+6
+7

Fig. 1 continued

cross-linked by one or more disulfide bridges. Two cysteine residues are located at 23 and 27 positions for 44
peptides in constant motif form CxxxxC. Some other cysteine residues are distributed in random positions in the
multiple alignment.
An unrooted phylogenetic tree of Gram-positive bacteriocins was constructed as shown in Fig. 2. Each branch or
sub-branch of the phylogenetic tree having a common
trunk forms a group or subgroup according to the proposed
structural classification and is then characterized by a
conserved consensus sequence motif. Computational
analysis has identified 12 distinct groups; each group is
characterized by one motif consensus.
Group 1 The sequence length of peptides belonging to
this group is comprised between 20 and 30 amino acid residues. Currently, 13 bacteriocins compose this group,
including mutacin 2, lacticin 481, bacteriocin J46, ruminococcin A, variacin, lacticin 3147, mersacidin, actargadine,
plantaricin W alpha, nukacin ISK1, mutacin H29B, streptococcin AFF22, and streptococcin AM49. This group corresponds to class I-Lantibiotic type A [3, 7] and type B.
Group 1 is characterized by the consensus motif GX3TX3EC
with 7.06 9 10-18 \ p-value \ 3.08 9 10-5 [2]. Two
subgroups are further characterized: subgroup 1a (lacticin
3147 A1, mersacidin, actargadine and plantaricin W alpha)
with the consensus motif GXXCTLXXEC (5.19 9 10-13 \
p-value \ 6.51 9 10-11) and subgroup 1b (Mutacin 2,
lacticin 481, bacteriocin J46, ruminococcin A, streptococcin

AFF22, streptococcin AM49, mutacin H29B, nukacin ISK1
and variacin) with the consensus motif GVX2TXSH/
YECX2NS/TW/FQ/AF/HV/LA/FTCC
(5.37 9 10-24 \
-18
p-value \ 1.25 9 10 ).
Group 2 is composed by duramycin C, ancovenin, and
cinnamycin. These bacteriocins belong to class I (Lantibiotics, type B) [27]. This group is characterized by the
following consensus motif: CX2SCSXGPXTX2CDGNTK
with a p-value comprised between 1.81 9 10-23 and
1.27 9 10-19.
Group 3 contains 29 bacteriocins with a sequence
length comprised between 36 and 50 residues of amino
acids. All members of class IIa are included in this group,
and were already characterized by a motif consensus.
Members of this group share a common consensus motif
KYYGNGVXCXKX2CXVD/NWX2A located at the Nterminal region, with a p-value [2] comprised between
3.06 9 10-22 and 1.73 9 10-16. All members of this
group have antilisterial activity [7] and can be further
subdivided into two subgroups:


Subgroup 3a: includes lactococcin MMFII, bacteriocin
31, enterocin SEK4, divergicin M35, enterocin A,
bavaricin MN, divercin V41, carnobacteriocin BM1,
piscicocin V1b, enterocin P, curvacin A and sakacin A.
The consensus motif YGNGVYCNX2KCWVX8I characterizes this subgroup with 1.36 9 10-23 \ p-value
\ 2.4 9 10-18.

123

A. Zouhir et al.

Fig. 2 Unrooted phylogenetic tree of Gram-positive bacteriocins. 3D
structure coordinates were obtained from the PDB (http://
www.rcsb.org/pdb). PDB accession ID numbers: Curvacin A: 2a2b;
Sakacin P: 1og7; Leucocin A: 3leu; Carnobacteriocin B2: 1cw5;

Nisin Z: 1wco; Cinnamycin: 2dde; Subtilosin A: 1pxq; Plnj: 2khf;
Actargadin: 1aj1. Pictures were generated using PyMOL software
(http://www.pymol.org). Alpha-helices and beta-sheets are shown in
red and purple, respectively



type A (class I) [7]. These peptides share the consensus
motif KATRX2TVSCK (7.65 9 10-11 \ p-value \ 1.44 9
10-10).
Group 6 is composed by cytolysin, plantaricin W beta,
and lacticin 3147 A2 which were set in class I (Lantibiotic:
Type A) [7]. This group is characterized by the consensus
motif SX3CPTTKC X3C (2.5 9 10-7 \ p-value \ 2.52 9
10-7).
Group 7 is composed of acidocin J 1132 beta and
plantaricin 1.25 beta which were classified in class IIb [22]
and class IId [3]. This group is identified by the consensus
sequence VX2CAS (5.08 9 10-10 \ p-value \ 8.63 9
10-10).
Group 8 is characterized by the consensus motif GX3
GGLX2IPXGPLXWXAGXAXV (1.09 9 19-9 \ p-value
\ 1.9 9 19-9). This group is composed of carnobacteriocin A and enterocin B which are classified in more than
one class including class IIc [31], class IId [21], and
unclassified bacteriocins [7].
Group 9 is composed of plantaricin S alpha, gassericin
T, and lacticin F. This group is characterized by the consensus motif CGPACX3GAHYXPIXWX2VTAATGGFGKIRK (1.17 9 10-34 \ p-value \ 2.08 9 10-34). These
bacteriocins were previously set in both class IIb [21] and
class IIe [31].

Subgroup 3b: encompasses carnobacteriocin B2, leucocin
C, listeriocin 743A, bavaricin A, sakacin P, mundticin,
enterocin CR35, mundticin KS, piscicolin 126, piscicocin
V1a, coagulin A, pediocin PA1, mesentericin Y105,
leucocin A, leucocin B Talla, plantaricin C19, and prebacteriocin 423. These peptides are secreted by ten species.
The common consensus which characterizes this subgroup
is KYYGNGVXCXKX2CXVXW with a p-value comprised between 1.14 9 10-14 and 4.95 9 10-11.

Group 4 is composed of eight bacteriocins (streptin,
epidermin, gallidermin, mutacin 1140, mutacin BNy 266,
subtilin, nisin A, and nisin Z) which are already classified in
class I (Lantibiotic: Type A) [7]. This group is characterized
by the consensus sequence SX3CTPGC (1.3 9 10-17 \
p-value \ 9.07 9 10-10). Sequence analysis revealed the
presence of two subgroups: 4a (subtilin, nisin A, nisin Z)
and 4b (streptin, epidermin, gallidermin, mutacin 1140,
mutacin BNy 266), which are characterized by the consensus motifs SXSLCTPGCXTGALX2CX3TXTCXI (2.84 9
10-36 \ p-value \ 4.72 9 10-25) and SX3CTPGCAXTGS
FNSYCC (5.41 9 10-14 \ p-value \ 6.45 9 10-6), respectively.
Group 5 Currently, two bacteriocins compose this group
(pep 5, epicidin 280) which were classified as lantibiotic

123

Classification of Gram-positive Bacteriocins

Group 10 is composed of the lacticin F lafX, acidocin
LF 221B and lactobin A which were classified in class IIb
[21, 22], class IId [3], class IIe [31] and as unclassified
bacteriocins [7]. These peptides share the consensus motif
GXWGX6GGAAXGGX2GY (1.04 9 10-25 \ p-value \
9.73 9 10-15).
Group 11 is composed of lactocin 705 and divergicin
750 which were set in the class IIb [7], class IIc [31] and
the class IId [3]. The consensus motif GX2GX2QX3
DFX2GX3GI characterize this group, with a p-value comprised between 1.67 9 10-7 and 4.94 9 10-7.
Group 12 includes lactococcin G beta, sublancin 168
and PlnJ, which were already set in the class IIb [7, 22] and
the class IIe [31]. This group is characterized by the motif
WX9GX3G (1.02 9 10-7 \ p-value \ 7.01 9 10-6).
As can be seen, the proposed classification scheme is
structure-based and permitted the identification of 12 distinct groups. Each group is represented by a distinct branch
on the phylogenetic tree and is characterized by a consensus sequence motif. As a result, more than 70% of the
studied Gram-positive bacteriocins are classified in groups
and subgroups. The presence of highly conserved motifs
among proteins of the same subgroup supports the phylogenetic relationships inferred from the bacteriocin
sequence alone. Conversely, few number of bacteriocin
sequences have a great evolutionary distances and consequently a high degree of sequence divergence. Thus, these
bacteriocins form unclear branches, with no obvious
sequence similarity (Fig. 1). This problem is due to the fact
that unclassified bacteriocins are not sufficiently studied
and sequenced to form a distinct branch and groups. This
structure-based classification of bacteriocins provides a
solution to current classification issues, as it groups bacteriocins based on their sequence similarities, thus avoiding
the presence of bacteriocins in more than one group at same
time. Considering the sequence structure as a single criterion, the proposed classification is in contrast with other
suggested classification schemes as some authors classified
the Gram-positives bacteriocins in three [7, 21, 23] or four
[17, 19] classes, based on multiple criteria which differ
from one author to another, as physicochemical proprieties,
number of cysteine residues, target organism, post-translational modifications, and mode of secretion. It is noteworthy
that only subclass IIa was characterized by a consensus
sequence motif [25]. However, the proposed classification
method extends such consensus motif-based technique to
cover 70% of the studied bacteriocins and provides a
solid methodology to classify new and yet to be classified
bacteriocins.
Until now it has not been easy to formulate a general and
coherent classification scheme that encompasses all of the
existing bacteriocins. In this paper, we revisited the previously proposed contradictory classification and we defined a

new structure-based sequence fingerprints that support a
subdivision of the bacteriocins’ family into 12 groups. The
paper lays down a resourceful and consistent classification
approach that resulted in classifying more than 70% of
bacteriocins known to date and with potential to identify
distinct classes for the remaining unclassified bacteriocins.
Unclassified bacteriocins and yet to be discovered ones are
expected to construct new distinct branches on the phylogenetic tree and form new groups. This structure-based
classification method lays down an adequate framework to
study the structure–function relationships and biological
aspects of bacteriocins. The picture that emerges from this
and others will improve our understanding of this promising
class of anti-infective agents, which are increasingly used in
various fields, including food preservation, food safety
applications and also for developing new drugs for medical
use.
Acknowledgments Authors thank Dr. Nefzi Adel and Dr. Sadok
Mokthar for their critical reading of the manuscript.

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