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Members of the Gram-positive bacterial genus Streptococcus are a diverse collection of species inhabiting many body sites and range from benign, nonpathogenic species to those causing life-threatening infections. The streptococci are also prolific producers of bacteriocins, which are ribosomally synthesized proteinaceous antibiotics that kill or inhibit species closely related to the producer bacterium. With the emergence of bacterial resistance to conventional antibiotics, there is an impetus to discover, and implement, new and preferably 'natural' antibiotics to treat or prevent bacterial infections, a niche that bacterial interference therapy mediated by bacteriocins could easily fill. This review focuses on describing the diversity of bacteriocins produced by streptococci and also puts forth a case for Streptococcus salivarius, a nonpathogenic and numerically predominant oral species, as an ideal candidate for development as the model probiotic for the oral cavity. S. salivarius is a safe species that not only produces broad-spectrum bacteriocins but harbors bacteriocin-encoding (and bacteriocin-inducing) transmissible DNA entities (megaplasmids).
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10.2217/FMB.09.61 © 2009 Future Med icine Ltd ISSN 1746- 0913
Future Microbiol. (20 09) 4( 7), x xx–x xx
part of
Future Microbiology
Streptococcal bacteriocins and
the case for Streptococcus
salivarius as model oral probiotics
Philip A Wescombe, Nicholas CK Heng, Jeremy P Burton, Chris N Chilcott
& John R Tagg
Author for correspondence: Departm ent of Microbiology and Immunology, University of Otago, PO Box 56,
Dunedin 9016, New Zealand n Tel.: +64 3479 7714 n Fax: +6 4 3479 8540 n john.tagg
Memb ers of the Gram- pos itive bacterial genu s Stre ptococcu s are a d iverse
collect ion of species inha b iti ng ma ny bo dy sites and ra nge from beni gn,
no npat hoge n ic sp e cie s to th ose ca using l ife - threat enin g inf ect ions . T he
streptococci are al so prolific producers of bacteriocins, which are ribosoma lly
synthesized proteinaceous antibiotics that kill or inhibit species closely related to
th e produc er b a ct eri u m. W ith t he em ergenc e of bacter ial resi s tan ce t o
conventional antibiotics, there i s a n impetu s to discover, and implement, new
and preferably ‘natural’ antibiotics to treat or prevent bacterial infections, a niche
that bacterial interference thera py mediated by bacteriocins could easily fill. This
review focuses on describing the diversity of bacteriocins produced by streptococci
and also puts forth a cas e for Streptococcus salivarius , a nonpathogenic and
numerica lly predominant oral species, as an ideal candidate for development
as the model prob iotic for the oral cavit y. S. sa livarius is a safe species that not
only produces broad- spectru m bacteriocins but harbors bacteriocin-encoding
(and ba cteriocin- inducing) trans mi ssible DNA entities (megaplasmids).
bacterial interferen ce
n bacteriocin n b acteriocin-
like inhibitory su bstance
n deferred antagonism
n lantibiotic n m egap lasm id
n oral microbiota n p robiotic
n salivarici n n Streptococcus
Historically, members of the genus Streptococcus,
together with those of the genus Staphylococcus,
were considered the prototypical Gram-positive
coccal pathogens of humans. Distinguished from
their staphylococcal cousins by their chain-form-
ing propensity, their lack of catalase and (gener-
ally) the absence of pigmentation, the streptococci
also display a marked preference for a mucosal
(especially oral) rather than a skin habitat.
Comprising a large and expanding compendium
of species, the streptococci officially belong to the
phylum Firmicutes, but their production of lactic
acid as the major end product of carbohydrate
metabolism qualifies them for membership of the
more familiar lactic acid bacteria group [1].
The streptococci include a number of patho-
genic or ‘rogue’ species, some of which can con-
tribute to the development of chronic conditions
such as dental caries or initiate acute infections
ranging from meningitis, pneumonia, endocardi-
tis and erysipelas to necrotizing fasciitis and occa-
sional delayed infection sequelae such as rheu-
matic fever and glomerulonephritis. The main
disease-causing streptococci are (generally) the
a-hemolytic Streptococcus mutans, Streptococcus
pneumoniae, Streptococcus mitis, Streptococcus
constellatus and Streptococcus sanguinis and, more
typically, the b-hemolytic Streptococcus pyogenes,
Streptococcus agalactiae and Streptococcus equisi-
milis. By contrast, other streptococci appear con-
sistently benign in their disposition to humans
and these reside peacefully as well-adapted
members of the normal commensal microbiota
of the oral cavity, the upper respiratory tract and
the intestine. Indeed, for commercial purposes,
some streptococci have been granted ‘generally
regarded as safe’ (GR AS) status in recognition
of their virtuous nature. Prime amongst these is
Streptococcus salivarius, which, while not yet hav-
ing GRAS status, is an oral species most closely
related to Streptococcus thermophilus, (formerly a
subspecies of S. salivarius), a bacterium exten-
sively used in the dairy industry for yoghurt
manufacture [1].
However, it is clear that amongst the various
streptococcal species all is not simply black or
white as far as their disease-causing propensity
is concerned. Indeed, members of all of the
so-called rogue species, when not flexing their
virulence attributes during aggressive forays,
commonly reside in a transient carriage state
within the oral commensal microbiota of healthy
humans. While held in check by other members
of the microbiota, these streptococci continue
to multiply in an orderly fashion, their covert
virulence traits undisclosed. Similarly, even the
most innocuous of commensal streptococci can
under some circumstances, such as in an immu-
nocompromised host, grow unfettered and to
menacing proportions capable of either directly
damaging the tissues or evoking unhelpful tissue
(immune) responses to their presence.
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Perspective Wescombe, Heng, Burton, Chilcott & Tagg
After more than half a century of using peni-
cillin and other therapeutic nonribosomally
synthesized antibiotics to control microbia l
infection, and with the development of bacte-
rial resistance to many of these antibiotics, there
is now an impetus to either enhance the kill-
ing spectra of existing synthetic antimicrobial
agents or discover new classes of antibiotics that
have novel modes of action, fewer side effects
and a lower tendency for resistance development.
Within the umbrella term ‘antibiotic’, the sub-
set known as ‘bacteriocins’ comprises the ribo-
somally synthesized proteinaceous compounds
released extracellularly by bacteria that can be
shown to kill, or interfere with the growth of,
other bacteria, generally closely related to the
producing bacterium. As yet uncharacterized
inhibitory agents that have been found to have
some bacteriocin-like properties are referred to
as bacteriocin-like inhibitory substances (BLIS).
The study of inter-bacterial inhibition can
trace its origins to Louis Pasteur who, in the
1870s, was seeking a way to control the growth
of the anthrax bacillus. He reported that the
growth of the bacillus was inhibited (both in vivo
and in vitro) when co-inoculated with ‘common
bacteria’ (probably Escherichia coli) isolated from
urine [2]. Pasteur’s pioneering studies led to sev-
eral decades of investigations that focused upon
the dosing of patients with relatively harmless
bacteria in an attempt to counter the prolifera-
tion of pathogensthe so-called ‘bacterial inter-
ference strategy’. Clearly, inter-species inhibition,
via bacterial interference mediated by ‘natural
antibiotics, such as bacteriocins, is a more desir-
able alternative to using synthetic antibiotic
agents, as they also cause relatively little collateral
killing of unrelated bacteria, principally deliv-
ering low concentrations of narrow spectrum
antimicrobials only to their immediate vicin-
ity. Therefore, bacteriocin-producing bacteria
offer (at least theoretically) a relatively targeted
approach to pathogen control.
In this article, we will describe the various
classes of bacteriocins produced by streptococci
and highlight specific examples that may be
potentially useful in controlling oral or medi-
cally relevant pathogenic species. Furthermore,
we propose that S. salivarius, the predominant
commensal streptococcus species in the human
oral cavity, may have an important role in main-
taining orderly population dynamics within oral
microbiota climax communities and that central
to this role is their ability to specifically com-
mission or decommission the production of dif-
fering combinations of bacteriocins encoded by
loci located on large extrachromosomal DNA
molecules (megaplasmids). The current scien-
tific basis for probiotic application of bacterio-
cin-producing S. salivarius to the control of oral
infections in humans is presented.
Streptococcal bacteriocins : how they
are detected & classified
In our laborator y, we test bacterial strains of
interest for their production of bacteriocins by
use of a standard three-step screening process:
nApplication of a deferred antagonism bacte-
riocin production (P)-typing procedure using
a set of nine standard indicator (detector)
strains [3,4];
nRepeating the bacteriocin P-typing procedure,
but incorporating a heating step (80°C for
45 min) prior to application of the indicator
nPCR-based detection of known bacteriocin-
related genes, usually of the lantibiotic (class I)
This three-step process can sometimes indi-
cate the production of multiple bacteriocins
by the test strain and may a lso give clues to
the possible class of inhibitory molecu le (s)
being produced. For example, the lantibiotics
typically produce heat-stable inhibition of the
Micrococcus luteus standard indicator strain,
wherea s inhibitory activity due to class III
(la rge) bacteriocins is invariably eliminated
by the heating step. Furthermore, the tried-
and-true deferred antagonism assay has shown
that even with the use of just a single set of
nine indicator strains, a very high frequency of
BLIS detection can be demonstrated. Among
the streptococci, S. mutans, S. salivarius and
S. uberis exhibit a particularly high incidence
of bacteriocin-producing capability, with some
strains producing combinations of bacterioc-
ins belonging to different classes. Some notable
examples include:
nBacteriocin-producing S. salivarius, some of
which display no less than five different bac-
te r i o ci n activit ie s [W esc omb e PA et  a l .,
U n publish edData];
nS. mutans UA140, which produces a lantibiotic
(mutacin I) and a class II inhibitory agent
(mutacin IV) [5];
nS. uberis 42, which produces the lantibiotic
nisin U, uberolysin (a circular bacteriocin)
and ubericin B (a class II peptide) [Heng NCK ,
Unpub lished Data] [6].
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Streptococcus salivarius as model oral probiotics Perspective
The bacteriocins of Gram-posit ive bac-
teria are a heterogeneous array of antibiot-
ics [7,8] , recently grouped into four classes [9]:
class Ithe lantibiotic peptides, class IIsmall
(<10 kDa) unmodified peptides, class IIIlarge
(>10 kDa) proteins and class IVthe circular
(cyclic) peptides. Examples of each class known
to be produced by streptococci are shown in
Figu r e 1
. The unifying characteristics of the
bacteriocins are their ribosomally synthesized
proteinaceous composition and their relatively
targeted killing of bacteria that are more closely
related to the producer bacterium. It has only
occasionally been observed that bacteriocins
may also exhibit toxicity for eukaryotic cells,
although in some instances the genetic loci
for bacteriocins may be closely linked, either
chromosoma lly or on plasmids, to bacterial
virulence determinants [10].
In our own studies we have been particularly
interested in the bacteriocin production and
sensitivity of S. salivarius and S. pyogenes, two
species that commonly cohabit the oral mucosa
and both of which occur exclusively in humans.
S. salivarius produce a particularly diverse array
of bacteriocins. Several of these strongly inhibit
S. pyogenes and other notorious upper respiratory
tract pathogens in vitro. Current knowledge of
a selection of streptococcal bacteriocins is sum-
marized below, while all known streptococcal
bacteriocins are briefly presented in
Tabl e 1
Class I : the lantibiotics
The term lantibiotic’ was coined to refer to
the diverse array of Gram-positive bacterial
antibiotic peptides that contain the nongeneti-
cally encoded amino acids lanthionine and/
or 3-met hyllanthionine, as well as various
other highly modified amino acids, commonly
including the 2,3-unsaturated amino acids
dehydroala nine and dehydrobutyrine. The
lantibiotics described to date, all of which are
produced exclusively by Gram-positive bacteria,
are initially produced as ribosomally synthesized
precursor peptides, which then undergo a series
of post-translational modifications to produce
the unusual amino acids that are intrinsic
components of the biologically active peptides.
Furthermore, lantibiotics are generally consid-
ered to predominantly, if not exclusively, act on
Gram-positive bacterial targets. As the family
of lantibiotic molecules grew, the individual
members were initially classified according to
the topology of their ring structures and their
biological activities as either type A (elongated
amphipathic structures) or type B (globular and
more compact structures) [11]. In order to encom-
pass the more recently described two-component
varieties, an additional type C was added to the
lantibiotic classification scheme
(Fig ure 1)
. The
type A lantibiotics are further divided into sub-
types A1 and A2 based on the size, charge and
sequence of their leader peptides
(Figu re 1)
. The
lantibiotics have been reviewed extensively over
the last decade and the reader is referred to some
of these accounts for a more complete overview
[12,13]. Here, we focus only on those lantibiotics
produced by S. salivarius, as other streptococcal
lantibiotics are produced by pathogenic species
such as S. pyogenes (streptin and SA-FF22) and
S. mutans (the mutacins)
(Figur e 1)
. Moreover, the
collective inhibitory spectra of S. salivarius lanti-
biotics encompass most, if not all, of the primary
bacterial targets in the oral cavity.
Salivaricin A
Salivaricin A is perhaps the most widely dis-
tributed of the streptococcal lantibiotics, being
produced by members of the species S. salivarius,
S. pyogenes, S. dysgalactiae and S. agalactiae. Six
subtypes of salivaricin A (A–A5; 2315–2368 Da)
have now been described, and each of these
peptides is capable of inducing production
of any one of the salivaricin A subtypes [14].
Salivaricin A inhibits the growth of most S. pyo-
genes when tested in vitro. Interestingly however,
nearly all S. pyogenes tested to date harbor the
salivaricin A1 structural gene, although strains
of only one serotype (M4) produce the biologi-
cally active salivaricin A1 molecule [15]. Other
S. pyogenes contain disrupted or truncated sali-
varicin A1 loci, which accounts for their inabil-
ity to produce the active bacteriocin [14]. This,
of course, begs the question of why the locus is
retained almost universally in S. pyogenes if its
bacteriocin product is not expressed. An answer
to this question may have been found in recent
studies that showed that the production of the
putative salivaricin A immunity protein SalY,
whose gene salY appears to have its own pro-
moter sequence, is important for the intracellu-
lar survival of S. pyogenes [16,17]. It was proposed
that SalY may function as a virulence factor for
S. pyogenes, conferring cross-protection against
the killing action of eukaryotic cationic pep-
tides. The implication is that, at least in S. pyo-
genes, the bacteriocin-negative, immune-positive
phenotype has been evolutionarily advantageous
and, thus, is over whelmingly conserved. Our
own (unpublished) studies have demonstrated
that SalY is also expressed by some indigenous
S. salivarius in the absence of expression of the
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Subtype A1
Nisin U
Mutacins I, III
Mutacin 1140
Bovicin HC5
Subtype A2
Salivaricins A, B, 9
Mutacin K8
Mutacin II
Type A
Class I
Type B
Type IIa
Ubericin A
Type IIb
Streptocin STH
Mutacin IV
Type IIc
Sanguinicin K11
Mutacins V, N
Class II
small (<10 kDa)
unmodified peptides
bacteriocins Class III
large (>10 kDa)
Class IV
cyclic peptides
Uberolysin Type IIIa
Zoocin A
Millericin B
Type IIIb
Type C
Perspective Wescombe, Heng, Burton, Chilcott & Tagg
Salivaricin B
Salivaricin B is the principal lantibiotic produced
by strain K12, the first commercially produced
probiotic S. salivarius (BLIS K12™ Throat
Guard, BLIS Technologies, Ltd). Salivaricin B
is a type A2, autoinducible lantibiotic with a
mass of 2740 Da, the production and immu-
nity determinants of which are present in an
eight-gene locus. Interestingly, the salivaricin B
locus generally appears to be located adjacent to
the salivaricin A2 locus on large megaplasmids
(of 19 salivaricin B-positive S. salivarius strains,
18 were also positive for salivaricin A) [21].
Salivaricin B is bacteriocidal in its action against
S. pyogenes and, in contrast to salivaricin A, has
not been found to be encoded by any S. pyogenes.
Moreover, no salivaricin B-resistant S. pyogenes
have yet been detected. Salivaricin B inhibits
S. sanguinis, S. equisimilis, S. agalactiae, S. pneu-
moniae, S. sobrinus, Corynebacterium diphtheriae,
Lactobacillus casei, Stomatococcus mucilagenosus
and Moraxella catarrhalis. This relatively broad
spectrum of activity is a distinctive feature of sal-
ivaricin B. Only a single variant of salivaricin B
has been detected, produced by a single strain
(SK648) of Streptococcus mitis. Comparison of
the salivaricin B structural gene (sboA) from
S. mitis SK648 with that of the wild-type K12
gene revealed a single point mutation resulting in
bioactive salivaricin A. However, in the case
of S. salivarius it appears more likely that the
expression of SalY in the absence of the homolo-
gous bacteriocin confers a survival advantage to
the host strain in the face of niche competition
from salivaricin A-producing S. salivarius, since
more than 10% of natural isolates of S. salivar-
ius are capable of producing salivaricin A [18].
Nevertheless, in view of the demonstrated con-
tribution of SalY to S. pyogenes intracellular sur-
vival, it will be of interest to determine whether
SalY expression confers a similar intracellular
survival capability upon S. salivarius.
An interesting feature of salivaricin A pro-
duction is that the active peptide, in addition
to being antibacterial, is also an autoinducing
signaling molecule that upregulates transcrip-
tion of the salivaricin A locus [19]. It is believed
that this mechanism allows the production of
salivaricin A by a S. salivarius population to
be significantly enhanced when there are large
enough numbers of bacteria to make it ecologi-
cally advantageous. This quorum-sensing-like
responsiveness could potentially be exploited to
provide additional protection to the human host
against S. pyogenes proliferation by using dietary
supplements of salivaricin A to boost salivari-
cin A production by populations of indigenous
salivaricin A-positive S. salivarius [20] .
Figure 1. Classification scheme for bacteriocins produced by streptococci (and other
Gram-positive bacteria) including examples of bacteriocins belonging to each class.
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Streptococcus salivarius as model oral probiotics Perspective
Table 1. Features, classification and probiotic potential of Streptococcal bacteriocins.
Bacteriocin Producing species Mass (Da) Classification Potential for probiotic use Ref.
Streptin 1
Streptin 2
Streptococcus salivarius/
Streptococcus pyogenes
I-A1 Has activity against S. pyogenes and other pathogens of the URT but is
also produced by some S. pyogenes
Mutacin III Streptococcus mutans 2266 I-A1 Pathogencause of dental caries [110]
Nisin U Streptococcus uberis 3029 I-A1 Usually isolated from animal sources (common cause of bovine mastitis)
and so may have little application for humans
Bovicin HC5 Streptococcus bovis 2440 I-A1 Pathogen – common cause of bacteremia and endocarditis [112]
Mutacin B-Ny266 S. mutans 2270 I-A1 Pathogencause of dental caries [113]
Mutacin 1140 S. mutans 2263 I-A1 Pathogencause of dental caries. However, a genetically modified
S. mutans strain producing this lantibiotic is under development by
Oragenics, Inc. ( FL, USA) for use in the prevention of dental caries
Mutacin I S. mutans 2364 I-A1 Pathogencause of dental caries [115]
Salivaricin A–A5 S. salivarius/S. pyogenes /
Streptococcus dysgalactiae/
Streptococcus agalactiae
2315–2368 I-A2 Currently produced by the probiotic S. salivarius K12 for halitosis, and
maintenance of throat health
Salivaricin B S. salivarius/
Streptococcus mitis
2740 I-A2 Currently produced by the probiotic S. salivarius K12 for halitosis and
maintenance of throat health
Salivaricin 9 S. salivarius 2560 I-A2 Has activity against S. pyogenes and other pathogens of the URT [Wescombe PA etal.,
Unpubli shed Data]
Salivaricin G32 S. salivarius 2667 I-A2 Has activity against S. pyogenes and other pathogens of the URT but is a
close homologue of SA-FF22 produced by S. pyogenes
Macedocin Streptococcus macedonicus 2795 I-A2 May be used in food fermentation processes; identical to SA-FF22 [24]
SA-FF22 S. pyogenes 2795 I-A2 Pathogencause of sore throats, scarlet fever, rheumatic fever and
Mutacin K8 S. mutans 2734 I-A2 Pathogencause of dental caries [118]
Mutacin II S. mutans 3245 I-A2 Pathogencause of dental caries [119]
Bovicin HJ50 S. bovis 3428 I-A2 (disulphide
Pathogen – common cause of bacteremia and endocarditis [120]
Smb S. mutans N.A. I-C Pathogencause of dental caries [121]
BHT-A Streptococcus
rattus/S. mutans
2802, 3375 I-C Pathogencause of dental caries [122]
N.A.: N ot applicable; URT: Upper respiratory tr act.
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Perspective Wescombe, Heng, Burton, Chilcott & Tagg
an arginine to histidine change at amino acid 44.
Moreover, the distribution of salivaricin B (less
than 1% of S. salivarius isolates) appears to be
markedly less than that of salivaricin A (~10%
of S. salivarius) [21].
The determinants for both salivaricin A and
salivaricin B (on the pSsalK12 megaplasmid)
appear to be transmissible between S. salivar-
ius strains with the megaplasmid having been
observed to move to another S. salivarius strain
in the oral cavity of subjects colonized with
strain K12 [22]. Oddly, the megaplasmid encod-
ing salivaricin A and salivaricin B appears to be
eliminated from the K12 strain at a frequency of
approximately 0.1% (one in 1000) when the host
bacterium is grown under conditions not con-
ducive to bacteriocin production. This indicates
that one or both of the bacteriocins act as selec-
tive agents for the retention of the megaplasmid,
further highlighting the ecological significance
of bacteriocin production in the oral cavity.
Salivaricin G32
Salivaricin G32 is a type A2 lantibiotic, the pro-
peptide form of which only differs from that of
the prototype S. pyogenes lantibiotic SA-FF22
(the first streptococcal type A2 lantibiotic to
be isolated) by the addition of a single lysine
amino acid residue at position 2 [23]. The spectra
of inhibitory activity of SA-FF22 and salivari-
cin G32 are similar and fairly narrow, that is,
restricted mainly to Lancefield group B, G, E
and L streptococci. Strains that produce either
SA-FF22 or salivaricin G32 produce the same
inhibitory profile (P-type) in deferred antago-
nism tests against the set of nine standard indi-
cators. SA-FF22 (and its variants) are widely
produced by streptococci, having been found in
S. pyogenes, S. dysgalactiae, S. macedonicus and
S. salivarius species to date. The lantibiotic pro-
duced by Streptococcus macedonicus, a bacterium
used in Greek cheese manufacturing, has been
designated ‘macedocin’ in order to avoid confu-
sion in the food industry over the safety of the
lantibiotic, even though the amino acid sequence
of the macedocin propeptide is identical to that
of SA-FF22 [24]. Salivaricin G32 has two copies
of the structural gene [23], mimicking the struc-
ture of the SA-FF22 locus in S. pyogenes serotype
M49 strains and that of the macedocin locus in
S. macedonicus [25,2 6]. S. pyogenes strains that pro-
duce SA-FF22 are resistant to salivaricin G32,
indicating that the SA-FF22 immunity genes
give cross-protection. Interestingly, while the
production of both SA-FF22 and salivaricin G32
is inducible and extracts from producers of each
Table 1. Features, classification and probiotic potential of Streptococcal bacteriocins (cont.).
Bacteriocin Producing species Mass (Da) Classification Potential for probiotic use Ref.
Ubericin A S. uberis 5271 II-A Pathogencause of bovine mastitis [31]
Streptocin STH Streptococcus gordonii N.A. II -B Not suitableb-hemolytic [33]
Sanguinicin K11 Streptococcus sanguinis 5070 II- C May provide lead structure for developing agents targeting S. agalactiae
or S. uberis
[Hale J DF etal., Unpubl ished
Zoocin A Streptococcus equi 27,000 III-A Pathogencause of equine strangles; bacteriocin is lytic for target
bacteria and may release cytotoxic /pyrogenic substances
Stellalysin Streptococcus constellatus 27,000 III-A Potential pathogencan cause abscesses [44]
Dysgalacticin Streptococcus dysgalactiae 21,493 III-B Pathogencauses sore throats. However, dysgalacticin is nonlytic and
may provide lead structure for developing agents targeting S. pyogenes
and related pathogens
SA-M57 Streptococcus pyogenes N.A. III-B Pathogencauses sore throat and sequelae such as glomerulonephritis.
However, SA-M57 is nonlytic and may provide lead structure for
developing agents targeting Listeria monocytogenes
Uberolysin S. uberis 7000 IV Pathogencauses bovine mastitis [6]
N.A.: N ot applicable; URT: Upper respiratory tr act.
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Streptococcus salivarius as model oral probiotics Perspective
lantibiotic can cross-induce production of the
other, the inducing peptide is not the active
lantibiotic itself [23]. Similar findings have been
reported for macedocin [26].
Salivaricin 9
Salivaricin 9 is a 2560 Da type A2 lantibiotic
produced by S. salivarius which appears to be
the agent responsible for an expanded SA-FF22-
like inhibitory spectrum in deferred antagonism
tests against the set of nine standard indica-
tors [Wescombe PA e tal., Unpublishe dData]. Unlike
SA-FF22, salivaricin 9 inhibits all S. pyogenes
strains tested to date, and is inhibitory towards
some enterococcal strains, but it does not appear
to inhibit many other pathogenic oral bacteria
such as S. mutans. The genetic determinants are
sometimes megaplasmid-associated and produc-
tion often seems to occur in conjunction with
salivaricin A. Like salivaricin A, it appears to
be autoinducible, although the exact nature of
the inducing molecule(s) has not been defined
and may differ from the lantibiotic peptide.
Interestingly, although it has many structural
similarities to SA-FF22, there does not appear
to be any cross-resistance between producers of
these two lantibiotics. Because of this, salivari-
cin 9 is an attractive candidate for control of
S. pyogenes.
Streptin is a type A1 lantibiotic (2445 Da) that
was originally isolated and characterized from
S. pyogenes [27,28]. It has a relatively broad spec-
trum of inhibitory activity, including all tested
streptococci from Lancefield groups A–G, L, M,
P, R–T, and also S. pneumoniae, S. salivarius and
S. uberis [29,30] . Many strains of S. mutans and
most of S. sanguinis are also sensitive to streptin.
The very broad inhibitory spectrum of streptin
makes it a very attractive lantibiotic for future
applications in human health. Interestingly,
although the structural gene for streptin has
been detected in a number of S. salivarius strains,
production of the active molecule occurs rela-
tively infrequently [31]. One potential reason for
this may be that the streptin genetic locus does
not encode a dedicated protease for the activa-
tion of the propeptide (via removal of the leader
sequence) and therefore requires a suitable host
protease for activation [28]. It is possible that
S. salivarius does not have a suitable protease or,
alternatively, the laboratory conditions used to
date may not be conducive for coexpression of
the protease together with the lantibiotic prepep-
tide. Nine of 38 S. salivarius producing strong
bacteriocin activity were found to have the
streptin structural gene, and it has been local-
ized to megaplasmids in at least some of these
strains [22]. However, many of these strains have
inhibitory spectra that are not consistent with
streptin being produced in significant concentra-
tions. Further investigations of P-type 777 S. sal-
ivarius strains having the streptin locus would
seem prudent considering the great potential
this molecule could have for use against a wide
variety of pathogens.
Class II: the small (<10 kDa)
unmodified peptides
Although not as numerous or as extensively
characterized as the lantibiotics, several non-
lantibiotic class II bacteriocins produced by oral
streptococci have been reported in the literature.
Class II represents the largest collection of bac-
teriocins as it encompasses all of the currently
known small (<10 kDa) unmodified (i.e., non-
lantibiotic and noncyclic) peptide bacteriocins
of Gram-positive bacteria [9]. To date, class II,
which is divided into three types
(Figu re
1), com-
prises over fifty members with diverse origins
ranging from genera inhabiting the oral cavity
and GI tract of humans (and other animals)
to species used as starter cultures in the dairy
and food industries. The class II peptides of
oral streptococci are only found as members of
types IIb (two-component) and IIIc (miscel-
laneous or nonpediocin-like), and they inhibit
their target organisms in both single-peptide
[31,32] and two-peptide forms; for example,
streptocin STH [33] . Interestingly, despite the
structural differences between streptococcal
class II members, and the obvious differences
between class II peptides and the lantibiotics,
they all share a common mode of export via
ATP-binding cassette (ABC) transporter com-
plexes. Like all of the lantibiotics, the known
streptococcal class II peptides are synthesized as
prepeptides with a signal peptide containing a
double-glycine (GG) motif. During export, the
signal peptide is cleaved by a peptidase domain
located at the N-terminus of the primary ABC
transporter protein. Whereas the streptococcal
class II bacteriocin-encoding loci, and similar
loci from most other Gram-positive organisms,
are organized such that the bacteriocin struc-
tural gene is adjacent to that of its dedicated
export system [31] , some unusual exceptions can
be found. For example, the multiple mutacins
produced by the S. mutans genome reference
strain UA159 are encoded by genes located
in disparate regions of the genome but are
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Perspective Wescombe, Heng, Burton, Chilcott & Tagg
exported by a single ABC transporter, NlmTE
[32]. Similarly, the loci encoding the bacteriocin/
hemolysin streptocin STH and its export system
in S. gordonii are located in different parts of
the chromosome. Interestingly, streptocin STH,
along with some S. mutans mutacins [34 ,35], is
under the control of the competence pathway,
which potentially allows these species to take
up and integrate exogenous DNA from the envi-
ronment (i.e., undergo genetic transformation)
[33,36]. As streptocin STH is a nonlytic inhibitory
agent, the function of the bacteriocin may be
to exclude potential competitor bacteria from
accessing the transient pool of available DNA
in the oral cavity. Conversely, the functions of
some of the mutacins, namely mutacin IV [34]
and CipB (also known as mutacin V [37]), appear
to be to lyse the target bacteria in order to release
a source of potentially useful DNA [34,35], or lyse
the producer cell itself (i.e., a ‘death protein’)
in times of stress in a quasialtruistic attempt to
provide a source of DNA to enhance the genetic
fitness of the bacterial community [38].
One of the more unusual class II bacteriocins
to be characterized is sanguinicin K11, a 5-kDa
extracellular class IIc bacteriocin produced by
S. sanguinis, one of the pioneer colonizers of the
human oral cavity [Hale JDF etal., Unpublished Data].
The inhibitory spectrum of sanguinicin K11 is
remarkable in that whilst most oral streptococci
are relatively unaffected, the peptide inhibits the
growth in vitro of all tested strains of the medi-
cally relevant S. agalactiae and S. uberis, one of
the principal bacterial causes of bovine mastitis.
Therefore, although sanguinicin K11 may have
limited application in controlling the overgrowth
of oral pathogens, it provides a lead structure
to facilitate development of new antimicrobial
agents targeting specific pathogenic bacteria.
Class III: the large
(>10 kDa) bacteriocins
While the majority of bacteriocins characterised
from streptococci are small (<10 kDa) peptides,
several large (>10 kDa) heat-labile antimicro-
bial proteins have been described at both the
biochemical and genetic level. The streptococ-
cal class III bacteriocins can be divided into
two distinct groups: class IIIa, the bacterio-
lytic enzymes (or bacteriolysins) that facilitate
the killing of sensitive strains by cell lysis, and
class IIIb, the nonlytic antimicrobial proteins [9].
To date, all streptococcal class III bacteriocins
have been shown to be secreted into the exter-
nal environment by the Sec-dependent secretory
pathway [36,39–42].
The first streptococcal class III bacteriocin
to be characterized was zoocin A, a 27-kDa
class IIIa domain-structured streptolytic pro-
tein produced by a strain of Streptococcus equi
subsp. zooepidemicus isolated from the oral cavity
[43]. Zoocin A specifically kills S. pyogenes and
S. mutans owing to its binding to and hydrolysis
of the peptidoglycan (cell wall) of these species
[39], thus making it an attractive candidate as a
control agent for the principal pathogenic oral
streptococci. However, the lytic nature of zoocin
A may preclude it from medical application as
potentially cytotoxic or pyrogenic intracellular
molecules, especially with S. pyogenes, may be
released into the external milieu upon cell lysis.
More recently, another zoocin A-like lytic bacte-
riocin, stellalysin, has been characterized from
Streptococcus constellatus [44] .
Dysgalacticin (21.5 kDa) and streptococcin
A-M57 (SA-M57; 17 kDa) are class IIIb bacterio-
cins produced by Streptococcus dysgalactiae subsp.
equisimilis and M-type 57 S. pyogenes, respec-
tively [4 0,41]. Although the predicted secondary
structures of both proteins are similar, their anti-
microbial spectra are remarkably different. The
antibacterial spectrum of dysgalacticin is fairly
narrow and is limited to strains of S. pyogenes
and S. dysgalactiae [40] . SA-M57, on the other
hand, inhibits an unusual range of organisms,
consisting mainly of nonstreptococcal Gram-
positive species such as Listeria spp. (including
Listeria monocytogenes) and Lactococcus lactis [41].
It has recently been demonstrated that dysga-
lacticin kills sensitive S. pyogenes cells in a novel
manner, presumably by targeting the glucose/
mannose sugar uptake system and disrupting the
proton motive force, leading to ATP starvation
and ultimately cell death [45]. Future mutational
studies with dysgalacticin and SA-M57 may lead
to the development of bacteriocin variants with
enhanced or expanded antimicrobial spectra.
Coupled with the advantage of their nonlytic
mode of action, bacteriocins of the dysgalac-
ticin group could be potentially useful inhibi-
tory agents against specific pathogenic bacterial
Class IV: the circular peptides
The fourth and arguably the most unique class
of bacteriocins produced by Gram-positive
bacteria is that encompassing the cyclic bacte-
(Figu re 1)
[9]. These inhibitory agents are
ribosomally-synthesized peptides but possess a
circular structure as they are post-translationally
processed such that the first and last amino acids
of the mature peptide are covalently bonded
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Streptococcus salivarius as model oral probiotics Perspective
the so-called head-to-tail ligation (reviewed
in [46]). To date, this unusual class of antimicro-
bial peptides comprises only a handful of mem-
bers, including a solitary streptococcal member
(uberolysin) produced by the bovine pathogen
S. uberis [6, 9].
Bacterial infections of the human oral
cavity potentially amenable to
probiotic control
Our own motivation for the development of an
oral probiotic was initially focused on developing
a natural alternative to the use of penicillin pro-
phylaxis to provide rheumatic fever patients with
protection against S. pyogenes infection. There is
still no immunization available to elicit immune
protection against streptococcal sore throat and
application of microbial interference poten-
tially offers a simple and inexpensive solution.
Upper respiratory tract infections are the most
common reason for an infant to visit a doctor.
While many of these infections are viral in ori-
gin, others involve bacterial disequilibria within
the indigenous microbiota. The most common
of the latter are otitis media, pharyngitis and
sinusitis, caused primarily by S. pneumoniae,
Haemophilus influenzae, Moraxella catarrhalis,
S. pyogenes and S. aureus.
Other all-too-commonly occurring imbal-
ances within the tongue or dental plaque
microbiotas can lead respectively to episodes of
halitosis and dental caries. In addition, in aging
populations, periodontal disease affects a large
proportion of the population and this is now
linked to the genesis of more serious systemic
diseases of, for example, the cardiovascular sys-
tem [47]. Hillman and associates have largely
focused their attentions upon the development
and application of bacterial interference to the
control of dental caries and the reader is referred
to some recent publications from that group for
more information [48–52]. The focus of the pres-
ent review, however, is upon the potential role
of nonpathogenic bacteriocin-producing S. sali-
varius to the control of oral infections other than
dental caries.
Pioneer observations of the potential
protection of the human host by
modulation of the commensal
oral microbiota
The contribution of inhibitor-producing com-
mensal streptococci to maintenance of upper
respiratory tract healt h was inferred from
the detection, in older participants, of a rela-
tively increased prevalence of indigenous oral
streptococci elaborating substances bacteri-
cidal for S. pyogenes, a nding that supports
the clinical observation that adults experience
fewer streptococcal infections than children
[53–57]. Furthermore, during outbreaks of strep-
tococcal tonsillitis, subjects having interfer-
ing a-hemolytic streptococci in their pharynx
appeared relatively resistant to streptococcal
tonsillitis [58,59]. Similarly, patients scheduled for
tonsillectomies had significantly fewer inhibitory
a-hemolytic streptococci in their oral microbiota
compared with the controls [59– 61]. Studies in
the USA [62] and Sweden [63–65] showed subjects
colonized with a-hemolytic streptococci had
fewer streptococcal throat infections. In follow-
up studies, treatment of streptococcal pharyn-
gitis with antibiotics followed by recolonization
with a-hemolytic streptococci seemed to reduce
the recurrence rate of streptococcal sore throat
[64, 66 ]. Studies of children in New Zealand
[67] and Brazil [68] showed that the occurrence
of S. pyogenes acquisitions correlated inversely
with naturally-occurring levels of S. salivarius-
producing BLIS molecules.
Studies comparing the inhibitory activity of
the normal nasopharyngeal microbiota against
common otitis media-causing pathogens in
healthy children, children with secretory otitis
media and children with recurrent otitis media
showed that the inhibitory activity of the nor-
mal bacterial microbiota against S. pneumoniae
and H. influenzae may be relatively low in some
children [69,70] . In a follow-up study, children
deemed susceptible to infection were recolo-
nized with a-hemolytic streptococci capable
of inhibiting otopathogens [64] . When exam-
ined 3 months later, 22 children (42%) given
the streptococcal spray were healthy and had
a normal tympanic membrane compared with
12 (22%) of those administered a placebo.
As early as 1985, S. salivarius strain TOVE-R
was shown to displace mutans streptococci from
the teeth of rats and inhibit caries development
[71–74]. More recently, antagonistic commensal
streptococci (including S. salivarius) were shown
to inhibit periodontal pathogens [75,76] . In an
animal model, inflammation and subgingival
recolonization by periodontal pathogens was
significantly reduced following treatment with
a mixture of three oral streptococci, including a
strain of S. salivarius [77]. Examination of the oral
microbiotas of subjects with halitosis revealed a
relative deficiency of S. salivarius [78], and coloni-
zation of participants suffering from severe halito-
sis with BLIS-producing S. salivarius significantly
reduced the level of oral malodor [79,80].
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Perspective Wescombe, Heng, Burton, Chilcott & Tagg
Oral probiotic attributes of S. salivarius
The normal oropharyngeal microbiota contains
a large number of native bacterial species, some
members of which exhibit in vitro inhibitory
activity against potential oropharyngeal patho-
gens. Included among the species able to prevent
biofilm formation or to positively influence peri-
odontal indices are Streptococcus oligofermentans
[81] , Streptococcus zooepidemicus and other uniden-
tified streptococcal spp. [82–84], Actinomyces spp.
[83], Weissella cibaria [85] and Veillonella spp. [86].
However, since most of these potentially antago-
nistic species are not predominant members of the
normal oral microbiota, careful consideration is
required before attempting to boost their num-
bers as a disease control measure.
Streptococcus salivarius populations are usu-
ally established in the mouth, nasopharynx and
intestinal tract within hours of birth [87–89] and
throughout life these bacteria remain as pre-
dominant streptococcal members of the normal
microbiota of the oral cavity and upper respira-
tory tract, especially on the papillary surface of
the tongue [78,89,90] . Depleted S. salivarius num-
bers and associated unbalanced overgrowth of
Candida or of oral anaerobes commonly occur
in maladies such oral thrush [91], halitosis [78] and
Sjogren’s syndrome [92].
Additional attributes of S. salivarius that sup-
port its high potential as an oral probiotic are its
excellent safety record and the fact that many
strains are prolific producers of a variety of bac-
teriocin activities (see above) [93,94]. Since human
saliva typically contains 1 × 107 S. salivarius
cells per ml, large numbers (~1 × 1010 cells) are
ingested daily. Breast-feeding infants consume
S. salivarius, as it is commonly present in human
breast milk [95–98]. Streptococcus salivarius, some-
times as the subspecies thermophilus, is also com-
monly present in starter cultures for traditional
supplements, fermented milks and raw-milk
European cheeses [99–105]. Owing to its strategic
location and dominant numbers, S. salivarius is
particularly well placed to perform a population
surveillance and management (i.e., ‘sentinel’)
role within the oral microbiota. This, we pro-
pose, is mediated via their production of anti-
competitor molecules, such as bacteriocins, and
the harboring of bacteriocin-encoding, trans-
missible, extrachromosomal DNA molecules (or
The megaplasmids of S. salivarius
As stated previously, many bacteriocin-produc-
ing S. salivarius harbor large extrachromosomal
megaplasmids of between 160 and 220 kbp in
size [22] . These megaplasmids have been shown
to contain most of the genetic loci encoding the
bacteriocin molecules produced by their host
strains. Moreover, the bacteriocin-encoding loci
are flanked by mobile genetic elements such as
insertion sequence (IS) elements [21]. The pres-
ence of such a wide variety of bacteriocin loci on
the megaplasmids (in some instances, up to four
different lantibiotics) indicates that the mega-
plasmids may function as ‘molecular reposito-
ries’ for S. salivarius bacteriocin determinants,
acquiring entire bacteriocin loci from a variety
of oral species via transposition of IS elements.
As the locus encoding salivaricin B has been
detected in strains of S. mitis without any appar-
ent flanking IS elements, it is therefore possible
that S. mitis may have been the original host for
the bacteriocin. The transmission of megaplas-
mids between different S. salivarius host cells has
been shown to occur in the oral cavity of humans
[22]. This mechanism may facilitate rapid and
flexible reassortments of potentially useful but
nonessential genetic loci within the consortium
of S. salivarius bacteria that characterize that
individual’s oral cavity. Whether intraspecies
megaplasmid transmission can occur is yet to
be established, although we have not yet been
able to detect any evidence of this occurring in
our studies of the oral streptococcal populations
of subjects colonized with megaplasmid-positive
S. salivarius strain K12 [Tagg JR Etal., Unpu blished
Data]. However, it is clear that much remains to
be elucidated regarding the fundamental biol-
ogy of S. salivarius megaplasmids. For example,
if the bacteriocin-related loci on a particular
megaplasmid comprise only 30% of the total
genetic complement, then what constitutes the
remaining 70%? It is tempting to speculate that
megaplasmids contain nonantibiotic-encoding
genetic determinants that confer on S. sali-
varius an ability to adapt more rapidly to and,
thus, survive extrinsic pressures imposed by, for
example, major dietary change, antibiotic or
antiseptic exposure or the transient introduction
of a foreign, potentially competitive bacterium.
Indeed, some recent results indicate that some
of the megaplasmids encode elements that foster
the attachment of S. salivarius to oral epithelial
cells [Tagg JR et al., Unpubli shed Data] . Moreover,
little is known about the precise molecular
mechanisms responsible for megaplasmid rep-
lication, the bacterial host range of S. salivarius
megaplasmids or the environmental conditions
that favor interstrain (or perhaps interspecies)
megaplasmid transfer. Whole-genome sequenc-
ing of megaplasmid-carrying S. salivarius strains
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Streptococcus salivarius as model oral probiotics Perspective
and ana lysis of the genome sequences is being
undertaken at the time of writing and may reveal
possible clues to these mechanisms.
Rationale for probiotic application of
S. salivarius
Practical applications of BLIS-producing S. sali-
varius as an agent of microbial interference are now
commercially available, for example, BLIS K12
Throat Guard, which features S. salivarius K12
as the active ingredient. This strain was initially
selected because of its strong in vitro BLIS activity
against Streptococcus pyogenes, the streptococcal
species most commonly implicated in bacterial
pharyngitis in westernized communities, and a
major cause of morbidity and mortality around
the world. Children naturally harboring S. sali-
varius that produces BLIS of similar types to those
of strain K12 have reduced levels of S. pyogenes
acquisition [68,9 4]. At present, S. pyogenes infec-
tions and their sequelae (especially rheumatic
fever) remain a major public health concern in
developing countries and in certain populations
in industrialized countries. In New Zealand and
Australia, these diseases particularly affect Maori,
Pacific Island and Aboriginal people, who have
among the highest reported rates of rheumatic
fever and severe invasive S. pyogenes diseases in
the world [106,107]. Worldwide, rheumatic fever
currently affects at least 12 million people and
some 400,000 die annually as a consequence of
rheumatic heart disease [201] .
Other approaches to deal with S. pyogenes
infections have included development of vaccines
and/or major screening and subsequent antibiotic
treatment regimens, all of which have so far failed
to come to fruition. We hypothesize that mega-
plasmid-containing S. salivarius populations act
to control and monitor not only S. pyogenes but
also other members of the bacterial microbiota
of the oral cavity in healthy individuals. Indeed,
some BLIS-producing S. salivarius could poten-
tially help control the onset of a wide variety of
oral infections ranging from streptococcal sore
throat and halitosis to gingivitis and oral thrush.
The surprisingly broad inhibitory activity of
some S. salivarius includes some microorganisms
(fungi and Gram-negative bacteria) not typically
targeted by bacteriocins of Gram-positive bac-
teria, Such findings significantly strengthen the
proposition that S. salivarius may become the
most important oral probiotic species.
From a future research perspective, the recently
demonstrated ability of some S. salivarius to
transfer their megaplasmids to other S. salivar-
ius, may facilitate the development of probiotic
S. salivarius that are equipped with bacteriocins
appropriate for the targeting of specific patho-
gens. Thus, a strain with good colonizing char-
acteristics (but inappropriate bacteriocinogenic
activity) may have its antimicrobial repertoire
modulated by incorporation of a BLIS-encoding
megaplasmid from a strongly bacteriocinogenic,
but relatively poorly colonizing strain.
At present, no single strain of S. salivarius
fulfils all the criteria of a ‘perfect probiotic’.
Desirable properties of probiotics include:
nLow pathogenic potential;
nPathogen-specific targeting;
nEase of propagation in vitro and of delivery in
a viable format to the human host;
nReliable integration within the host i ndigenous
nEase of elimination, if required, from the
human host (e.g., with antibiotic dosing);
nGenetic stability ;
nPersistenc e at ef fective levels within a
c hosen habitat.
As the antithesis to these ideal characteristics
of a probiotic, the reader may question whether
there may also be risks or potential shortcom-
ings associated with probiotic usage:
nCandidate probiotics should be rigorously eval-
uated for their in vitro and in vivo safety on a
strain-by-strain basis [108,109]. Even so, in immu-
nocompromised individuals, any commensal
species has the propensity to cause infections;
nThe targeting of most bacteriocins produced
by Gram-positive bacteria is not species
s pecific. Therefore, the introduction of large
numbers of a bacteriocin-producing strain
would be anticipated to cause some modula-
tion of the composition of the indigenous
microbiota of the host, in addition to enhanc-
ing the exclusion of target pathogens. In the
case of probiotic S. salivarius, it is known that
approximately 5% of humans naturally harbor
large populations of S. salivarius, with BLIS
repertoires similar to that of strain K12 and,
therefore, the presence of this bacterium is
consistent with the occurrence of a ‘natural’
oral microbiota;
nThe shelf life of probiotics is being improved
and assured by improvements in commercial
prac tice and more rigorou s regulator y
s tandards. Interestingly, some of the benefits
especially relating to immune enhancement,
can also be obtained from nonviable p robiotic
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Perspective Wescombe, Heng, Burton, Chilcott & Tagg
nIt is known that not all individuals colonize
effectively with a standardized regimen of pro-
biotic application. This is owing to differences
in the competitive capabilities of the host’s
existing microbiota. In such cases, more fre-
quent dosing may be required to induce a per-
sistent presence of the probiotic or to maintain
its beneficial physiological response in the host.
This may be less important in cases where the
benefit of the probiotic, such as transient stim-
ulation of innate immunity, can be achieved
with short-term exposure;
nThe elimination of the probiotic from the
host microbiota, if deemed necessary, should
be facilitated by ensuring that these strains
do not carry resista nc e determina nts to
c ommonly used antibiotics;
nThe flexible content of megaplasmid DNA in
S. salivarius is not consistent with stability of
the plasmid-located determinants and so it will
be important to continue to monitor the con-
tent of these plasmids. To date, there is no
evidence for the presence of streptococcal
virulence determinants on these plasmids;
nThe extent of persistence of the probiotics at
effective levels within the host microbiota will
vary from host to host. To help overcome
uncertainties relating to this, it is recommended
that probiotic doses are taken regularly and
especially at times of perceived infection risk.
In the future, the development of simple, inex-
pensive screening tests to monitor levels of the
probiotic within the host microbiota will help
to alleviate much of this concern.
Executive summary
The genus Streptococcus
nA family of Gram-positive bacteria that produce lactic acid as the primary product of fermentation.
nMany streptococcal species reside in the oral cavity of humans and other animals.
nMany species, such as Streptococcus salivarius and Streptococcus sanguinis, are primary colonizers and/or benign inhabitants.
nSome species cause infections, such as dental caries (Streptococcus mutans), pharyngitis and rheumatic fever (Streptococcus pyogenes).
Streptococcal bacteriocins: detection & diversity
nBacteriocins are ribosomally synthesized antimicrobial proteins produced by bacteria that kill species related to the producer bacterium.
nStreptococci are prolific producers of bacteriocins and inhibitory activity is detected, and possibly identified, by several standard procedures:
– An agar-based deferred antagonism assay (production [P] -typing) is the primary and most commonly used screening method
employing a set of nine standard indicator strains.
– Other discriminating tests include thermal stability, for example, heating to 80°C, and molecular techniques, for example, PCR-based
screening for known generic bacteriocin-related genes.
– As yet uncharacterized inhibitory agents that have been found to have some bacteriocin-like properties are referred to as
bacteriocin-like inhibitory substances (BLIS).
Streptococcus salivarius bacteriocins are primarily lantibiotics
nSalivaricin A: bacteriostatic mode of action comprising six subtypes and produced by four streptococcal species. SalY, the salivaricin A
immunity protein is widely distributed in S. pyogenes and may provide cross-protection against eukaryotic cationic peptides.
nSalivaricin B: bacteriocidal broad spectrum peptide. Salivaricin A and salivaricin B loci are typically located adjacent to each other in
transmissible large extrachromosomal DNA molecules (megaplasmids).
nSalivaricin G32: very similar, but not identical, to the first streptococcal lantibiotic (SA-FF22) ever described. Inhibitory spectrum is
mainly limited to b-hemolytic streptococci. SA- FF22 has been given ‘food grade’ status in the form of macedocin (produced by
Streptococcus macedonicus).
nSalivaricin 9: exhibits an expanded salivaricin G32-like inhibitory spectrum and its biosynthetic locus may be megaplasmid-borne.
nStreptin: a very broad-spectrum lantibiotic also produced by S. pyogenes.
Desired characteristics of ‘the perfect probiotic
nThe perfect probiotic strain should be of low pathogenic potential, pathogen-specific in its targeting, easily propagated in vitro and readily
delivered in a viable format to the human host, reliably integrated within the host indigenous microbiota, readily eliminated, if required,
from the human host (e.g., with antibiotic dosing), genetically stable and capable of persisting at effective levels within a chosen habitat.
The case for Streptococcus salivarius as a model oral probiotic
nOne of the primary colonizers of the human oral cavity and persists as a dominant bacterial species throughout the life of its human host.
nHas never been associated with infections in healthy humans.
nHigh naturally occurring levels of S. salivarius are correlated with lower incidence of S. pyogenes acquisition.
nMany strains produce bacteriocins and BLIS that inhibit (in vitro) a variety of oral pathogens.
nSome S. salivarius harbor megaplasmids, which have the potential of enhancing the antimicrobial repertoire of indigenous strains
through horizontal gene transfer.
nSome strains have excellent safety records and are commercially available as bacterial replacement therapy preparations.
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Streptococcus salivarius as model oral probiotics Perspective
Tailor-made probiotics potentia lly offer a
natural approach to infection control. The
strategy would be cost effective, easily imple-
mented and not fraught with many of the
adverse complications associated with vacci-
nation (hypersensitivit y, cross-reactivity) or
chemotherapy (resistance development, hyper-
sensitivity, toxic reactions).
Future perspective
The probiotics field is now expanding rapidly,
with both scientists and the genera l public
becoming more acutely aware of the potential
beneficial outcomes to be derived by fostering
the interaction of certain nonpathogenic bac-
teria with the human host. To date, the major-
ity of probiotics have been derived from and
are designed for use in the GI tract. As these
have proven to be both medically and com-
mercially successful, the application of these
strains to other areas of the human body has
become attractive. Indeed, most attempts to
apply probiotics to nonintestinal surfaces have,
rather anomalously, utilized strains of intesti-
nal origin. Owing to the tissue specificity of
most bacteria, it seems that these gut-derived
strains are unlikely to be optimally effective in
foreign terrain and more successful probiotic
candidates would be expected to be derived
from those body sites where they are intended
to be used. As appreciation of this notion
spreads within the research communit y, we
expect t here to be a marked increase in the
number of probiotics of novel species that are
eva luated for application to body sites other
than the GI tract.
It is becoming increasingly clear that the opti-
mal time for implanting beneficial microorgan-
isms is in the first few years of life, when there
is a window of opportunity for influencing the
composition of the indigenous microbiota. We
predict that various combinations of indigenous
microbes will be assembled and proposed for
use to seed the various accessible human body
surfaces and that these bacterial combinations
may even contain genetically modified organ-
isms as these become more widely acceptable to
the public. The attempts by microbial ecologists
to formulate ‘optimal bacterial colonization
consortia will doubtless become more informed
and will accelerate as a consequence of the rapid
technological advances of the postgenomic era.
Financial & competing interests disclosure
Philip A Wescombe, Jeremy P Burton , Chris N
Chilcott & John R Tagg are employed by BLIS
Technologies Ltd, which is a company that specializes
in the development and production of probiotic micro-
organisms including Streptococcus salivarius K12.
The authors have no other relevant affiliations or
financial involvement with any organization or entity
with a financial interest in or financial conflict with
the subject matter or materials discussed in the manu-
script apart from those disclosed.
No writing assistance was utilized in the production
of this manuscript.
Papers of special note have been highlighted as:
n of interest
nn of considerable interest
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streptococci: overview of taxonomic and
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Author Proof
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n Philip A Wescombe
BLIS Technologies Ltd, Centre for
Innovation, University of Otago,
PO Box 56, Dunedin 9016, New Zealand.
Tel.: +64 3479 5337
Fax: + 64 3479 8554
n Nicholas CK Heng
Department of Oral Sciences, Faculty of
Dentistr y, University of Otago,
PO Box 647, Dunedin 9054, New Zealand
Tel.: +64 3479 9254
Fax: + 64 3479 7078
n Jeremy P Burton
BLIS Technologies Ltd, Centre for
Innovation, University of Otago,
PO Box 56, Dunedin 9016, New Zealand
Tel.: +64 3479 5337
Fax: + 64 3479 8554
n Chris N Chilcott
BLIS Technologies Ltd, Centre for
Innovation, University of Otago,
PO Box 56, Dunedin 9016, New Zealand
Tel.: +64 3479 5337
Fax: + 64 3479 8554
n John R Tagg
Department of Microbiology and
Immunology, University of Otago,
PO Box 56, Dunedin 9016, New Zealand
Tel.: +64 3479 7714
Fax: + 64 3479 8540
... 4. Стабильность при хранении [4,10]. Перечисленным требованиям отвечает вид Streptococcus salivarius, который был выбран для изучения как оральный пробиотик за имеющиеся потенциальные эффекты для здоровья [11,12]. Streptococcus salivarius является грамположительным бактериальным комменсалом, он одним из первых колонизирует полость рта человека и сохраняется в качестве доминирующего представителя нормальной микробиоты в течение жизни. ...
... Декстраназа расщепляет декстран, что способствует снижению образования зубного налета. Уреаза катализирует гидролиз мочевины до аммиака и углекислого газа, это обеспечивает буферные свойства, которые защищают среду от низкого pH и способствуют предотвращению деминерализации эмали [11][12][13]17]. Безопасность этого штамма подтверждена агентством FDA (U.S. Food and Drug Administration), которое Оригинальная статья І Original article присвоило Str. ...
... The science of probiotics using non-pathogenic oral bacteria, such as Streptococcus Salivarius M18, creates strain-specific and tissue-mediated antagonism by releasing bacteriocins (salivaricins) with antibiotic action [64,65]. A literature review showed that using probiotics regularly reduced the risk of caries with inhibitory action on cariogenic bacteria by enhancing the concentration of commensal bacteria in the oral cavity [66,67]. The potential of the probiotic on the attendance and endurance of S. salivarius and its ability to produce bacteriocins appears to be concentration-dependent, which is dose-dependent [64]. ...
... Streptococcus salivarius is present in greater amounts on the tongue's surface. The release of bacteriocins by some bacterial strains would eliminate pathogenic bacteria [66,[70][71][72]. Molecular studies have shown that these megaplasmids can synthesize molecules promoting host cell adhesion without generating antibiotic resistance [73]. ...
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The oral microbiota plays a vital role in the human microbiome and oral health. Imbalances between microbes and their hosts can lead to oral and systemic disorders such as diabetes or cardio-vascular disease. The purpose of this review is to investigate the literature evidence of oral microbiota dysbiosis on oral health and discuss current knowledge and emerging mechanisms governing oral polymicrobial synergy and dysbiosis; both have enhanced our understanding of pathogenic mechanisms and aided the design of innovative therapeutic approaches as ORALBIOTICA for oral diseases such as demineralization. PubMed, Web of Science, Google Scholar, Scopus, Cochrane Library, EMBEDDED, Dentistry & Oral Sciences Source via EBSCO, APA PsycINFO, APA PsyArticles, and [email protected] were searched for publications that matched our topic from January 2017 to 22 April 2022, with an English language constraint using the following Boolean keywords: (“microbio*” and “demineralization*”) AND (“oral microbiota” and “demineralization”). Twenty-two studies were included for qualitative analysis. As seen by the studies included in this review, the balance of the microbiota is unstable and influenced by oral hygiene, the presence of orthodontic devices in the.
... In particular, preliminary studies investigated the potential of one streptococcal strain, Streptococcus salivarius 24SMB. S salivarius is a non-pathogenic species able to colonize the oral cavity and can be considered a primary BLIS producer [37]. This strain exerted significant activity against S pneumoniae, was harmless to other S salivarius species, was non-pathogenic, and adhered to human larynx cells [38]. ...
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Recurrent respiratory infections (RRIs) account for relevant economic and social implications and significantly affect family life. Local Bacteriotherapy (LB) represents an innovative option in preventing RRIs. Local bacteriotherapy consists of administering “good” and safe bacteria (probiotics) by nasal or oral route. In particular, two strains (Streptococcus salivarius 24SMB and Streptococcus oralis 89a) are commonly used. The present article presents and discusses the literature concerning LB. Infections of airways include the upper and lower respiratory tract. A series of clinical trials investigated the preventive role of LB in preventing upper and lower RIs. These studies demonstrated that LB safely reduced the prevalence and severity of RIs, the use of antibiotics, and absences from school. Therefore, Local Bacteriotherapy may be considered an interesting therapeutic option in RRI prevention.
... Notably, Lactobacillus plantarum DR7, isolated from bovine milk, protected cattle against upper respiratory tract infections [27]. The bacteriocins produced by several members of the Streptococcus genus could act as "natural" antibiotics to treat or prevent bacterial infections [28]. Evidently, a reduced abundance of members of Pasteurellales and Lactobacillales may also contribute to the susceptibility of bovines to respiratory disease. ...
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Bovine respiratory disease (BRD) continues to pose a serious threat to the cattle industry, resulting in substantial economic losses. As a multifactorial disease, pathogen infection and respiratory microbial imbalance are important causative factors in the occurrence and development of BRD. Integrative analyses of 16S rRNA sequencing and metabolomics allow comprehensive identification of the changes in microbiota and metabolism associated with BRD, making it possible to determine which pathogens are responsible for the disease and to develop new therapeutic strategies. In our study, 16S rRNA sequencing and metagenomic analysis were used to describe and compare the composition and diversity of nasal microbes in healthy cattle and cattle with BRD from different farms in Yinchuan, Ningxia, China. We found a significant difference in nasal microbial diversity between diseased and healthy bovines; notably, the relative abundance of Mycoplasma bovis and Pasteurella increased. This indicated that the composition of the microbial community had changed in diseased bovines compared with healthy ones. The data also strongly suggested that the reduced relative abundance of probiotics, including Pasteurellales and Lactobacillales, in diseased samples contributes to the susceptibility to bovine respiratory disease. Furthermore, serum metabolomic analysis showed altered concentrations of metabolites in BRD and that a significant decrease in lactic acid and sarcosine may impair the ability of bovines to generate energy and an immune response to pathogenic bacteria. Based on the correlation analysis between microbial diversity and the metabolome, lactic acid (2TMS) was positively correlated with Gammaproteobacteria and Bacilli and negatively correlated with Mollicutes. In summary, microbial communities and serum metabolites in BRD were characterized by integrative analysis. This study provides a reference for monitoring biomarkers of BRD, which will be critical for the prevention and treatment of BRD in the future.
... Commensal microbiota play pivotal role in maintaining oral and systemic health; as local and systemic immunity develop in response to interaction between host immune system and commensal flora 12 . Presence of oral microbiota in the mouth cavity prevents colonization by pathogenic organisms, a condition known as colonization resistance 13 , also some strains of streptococcus salivaris produce bacteriocin which is a protein inhibiting growth of gram negative bacteria linked to periodontitis 14 . Another interesting role of oral microbiome is related to metabolism of ingested nitrate that is reduced by oral flora into nitrite that is subsequently absorbed into bloodstream and converted into nitric oxide that is important for cardiovascular health 15 . ...
... As reported in the literature, they are known to produce bacteriocins, which are ribosomally synthesized antimicrobials that typically have a narrow inhibitory spectrum directed against relatively closely related bacteria. 25 M18 also releases salivaricin M, which limits the growth of caries-causing bacterial species, S. mutans and S. sobrinus. 26 Plaque index (Silness and Loe, 1964) measures the thickness of plaque on the gingival one-third. ...
... S. salivarius M18 produces urease and dextranase enzymes that neutralize the acidity of saliva and inhibit the microorganisms present in oral biofilms. Streptococcus oligofermentans has shown strong adhesion and a low capacity to metabolize carbohydrates but can produce H 2 O 2 to inhibit pathogens such as S. mutans [115,116]. ...
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Bacteria in the oral cavity, including commensals and opportunistic pathogens, are organized into highly specialized sessile communities, coexisting in homeostasis with the host under healthy conditions. A dysbiotic environment during biofilm evolution, however, allows opportunistic pathogens to become the dominant species at caries-affected sites at the expense of health-associated taxa. Combining tooth brushing with dentifrices or rinses combat the onset of caries by partially removes plaque, but resulting in the biofilm remaining in an immature state with undesirables’ consequences on homeostasis and oral ecosystem. This leads to the need for therapeutic pathways that focus on preserving balance in the oral microbiota and applying strategies to combat caries by maintaining biofilm integrity and homeostasis during the rapid phase of supragingival plaque formation. Adhesion, nutrition, and communication are fundamental in this phase in which the bacteria that have survived these adverse conditions rebuild and reorganize the biofilm, and are considered targets for designing preventive strategies to guide the biofilm towards a composition compatible with health. The present review summarizes the most important advances and future prospects for therapies based on the maintenance of biofilm integrity and homeostasis as a preventive measure of dysbiosis focused on these three key factors during the rapid phase of plaque formation.
... Meanwhile, members of the Streptococcus genus are anciently known to cause life-threatening infections. However, recent studies have suggested the potential probiotic uses of some species such as S. salivarius [83] and S. thermophilus [84]. ...
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Functional symbiotic intestinal microbiota regulates immune defense and the metabolic processing of xenobiotics in the host. The aryl hydrocarbon receptor (AhR) is one of the transcription factors mediating host–microbe interaction. An in vitro static simulation of the human colon was used in this work to analyze the evolution of bacterial populations, the microbial metabolic output, and the potential induction of AhR transcriptional activity in healthy gut ecosystems. Fifteen target taxa were explored by qPCR, and the metabolic content was chromatographically profiled using SPME-GC-MS and UPLC-FLD to quantify short-chain fatty acids (SCFA) and biogenic amines, respectively. Over 72 h of fermentation, the microbiota and most produced metabolites remained stable. Fermentation supernatant induced AhR transcription in two of the three reporter gene cell lines (T47D, HepG2, HT29) evaluated. Mammary and intestinal cells were more sensitive to microbiota metabolic production, which showed greater AhR agonism than the 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) used as a positive control. Some of the SCFA and biogenic amines identified could crucially contribute to the potent AhR induction of the fermentation products. As a fundamental pathway mediating human intestinal homeostasis and as a sensor for several microbial metabolites, AhR activation might be a useful endpoint to include in studies of the gut microbiota.
The human microbiome is composed of a collection of dynamic microbial communities that inhabit various anatomical locations in the body. Accordingly, the coevolution of the microbiome with the host has resulted in these communities playing a profound role in promoting human health. Consequently, perturbations in the human microbiome can cause or exacerbate several diseases. In this Review, we present our current understanding of the relationship between human health and disease development, focusing on the microbiomes found across the digestive, respiratory, urinary, and reproductive systems as well as the skin. We further discuss various strategies by which the composition and function of the human microbiome can be modulated to exert a therapeutic effect on the host. Finally, we examine technologies such as multiomics approaches and cellular reprogramming of microbes that can enable significant advancements in microbiome research and engineering.
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The levels of several microbial groups of aerobic mesophilic flora, aerobic psychrotrophic flora, lactic acid bacteria, Micrococcaceae, enterococci, Enterobacteriaceae, and molds and yeasts were investigated during the manufacture of fresh white cheese of a Serbian craft variety without the addition of starter culture. This variety of cheese is made in farmhouses from cow, sheep and goat's milk. White fresh cheese from mountain villages of Serbia has economical importance for this area. The study of the microbial characteristics of this cheese constitutes the first step towards the establishment of a starter culture which would allow the making of a product both more uniform and safer. The total microbial counts were high in these variety of cheeses. Almost all the microbial groups reached their maximum counts in curd. Lactic acid bacteria were the major microbial group, reaching count similar to the total aerobic mesophilic flora at all sampling points. Lactococcus lactis subsp. lactis dominated in milk (62,5%) of the isolates obtained in the Man Rogosa Sharpe (MRS) agar at these sampling points, while the Lactobacillus casei subs.casei was the most predominant species (83,5% of isolates obtained at these sampling points). The purpose of this study was to investigate the microflora of white cheeses with special emphasis on the autochthonous lactic acid bacteria involved in fermentation of this cheeses depending on the geographical location where the cheeses were manufactured.
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This paper reports the characterisation of a bacteriocin-like inhibitory substance (BLIS), zoocin A, produced by Streptococcus zooepidemicus strain 4881 that exhibits inhibitory activity against strains of S. mutans, S. sobrinus and S. cricetus. A study of the inhibitory activity of strain 4881 against 98 bacterial strains using a deferred antagonism procedure showed that S. mutans strains were relatively more sensitive than were strains of S. salivarius and S. sanguis. Zoocin A was purified from the supernate fluid of chemically defined medium cultures of strain 4881. Purified zoocin A (a protein of estimated MW 30 000) retained biological activity over a wide pH range (4–10), was sensitive to several proteolytic enzymes and was relatively stable at 37°C but not at 60°C. A triple-species (S. mutans, S. sanguis and Actinomyces viscosus) plaque model was used to test the ability of zoocin A to modify plaque composition. Brief (2 min) exposure of preformed plaque to zoocin A resulted in a significant decrease in the proportion of S. mutans and this effect persisted for up to 20 h after treatment.
In recent years, a group of antibacterial proteins produced by gram-positive bacteria have attracted great interest in their potential use as food preservatives and as antibacterial agents to combat certain infections due to gram-positive pathogenic bacteria. They are ribosomally synthesized peptides of 30 to less than 60 amino acids, with a narrow to wide antibacterial spectrum against gram-positive bacteria; the antibacterial property is heat stable, and a producer strain displays a degree of specific self-protection against its own antibacterial peptide. In many respects, these proteins are quite different from the colicins and other bacteriocins produced by gram-negative bacteria, yet customarily they also are grouped as bacteriocins. Although a large number of these bacteriocins (or bacteriocin-like inhibitory substances) have been reported, only a few have been studied in detail for their mode of action, amino acid sequence, genetic characteristics, and biosynthesis mechanisms. Nevertheless, in general, they appear to be translated as inactive prepeptides containing an N-terminal leader sequence and a C-terminal propeptide component. During posttranslational modifications, the leader peptide is removed. In addition, depending on the particular type, some amino acids in the propeptide components may undergo either dehydration and thioether ring formation to produce lanthionine and beta-methyl lanthionine (as in lantibiotics) or thio ester ring formation to form cystine (as in thiolbiotics). Some of these steps, as well as the translocation of the molecules through the cytoplasmic membrane and producer self-protection against the homologous bacteriocin, are mediated through specific proteins (enzymes). Limited genetic studies have shown that the structural gene for such a bacteriocin and the genes encoding proteins associated with immunity, translocation, and processing are present in a cluster in either a plasmid, the chromosome, or a transposon. Following posttranslational modification and depending on the pH, the molecules may either be released into the environment or remain bound to the cell wall. The antibacterial action against a sensitive cell of a gram-positive strain is produced principally by destabilization of membrane functions. Under certain conditions, gram-negative bacterial cells can also be sensitive to some of these molecules. By application of site-specific mutagenesis, bacteriocin variants which may differ in their antimicrobial spectrum and physicochemical characteristics can be produced. Research activity in this field has grown remarkably but sometimes with an undisciplined regard for conformity in the definition, naming, and categorization of these molecules and their genetic effectors. Some suggestions for improved standardization of nomenclature are offered.
The complex ecosystem of intestinal microflora is estimated to harbor approximately 400 different microbial species, mostly bacteria. However, studies on bacterial colonization have mostly been based on culturing methods, which only detect a small fraction of the whole microbiotic ecosystem of the gut. To clarify the initial acquisition and subsequent colonization of bacteria in an infant within the few days after birth, phylogenetic analysis was performed using 16S rDNA sequences from the DNA isolated from feces on the 1st, 3rd, and 6th day. 16S rDNA libraries were constructed with the amplicons of PCR conditions at 30 cycles and 50 C annealing temperature. Nine independent libraries were produced by the application of three sets of primers (set A, set B, and set C) combined with three fecal samples for day 1, day 3, and day 6 of life. Approximately 220 clones (76.7%) of all 325 isolated clones were characterized as known species, while other 105 clones (32.3%) were characterized as unknown species. The library clone with set A universal primers amplifying 350 by displayed increased diversity by days. Thus, set A primers were better suited for this type of molecular ecological analysis. On the first day of the life of the infant, Enterobacter, Lactococcus lactic, Leuconostoc citreum, and Streptococcus mitis were present. The largest taxonomic group was L. lactic. On the third day of the life of the infant, Enterobacter, Enterococcus faecalis, Escherichia coli, S. mitis, and Streptococcus salivarius were present. On the sixth day of the life of the infant, Citrobacter, Clostridium difficile, Enterobacter sp., Enterobacter cloacae, and E. coli were present. The largest taxonomic group was E. coli. These results showed that microbiotic diversity changes very rapidly in the few days after birth, and the acquisition of unculturable bacteria expanded rapidly after the third day.
A bacteriocin-producing strain was isolated from raw milk and named Streptococcus bovis HJ50. Like most bacteriocins produced by lactic acid bacteria, bovicin HJ50 showed a narrow range of inhibiting activity. It was sensitive to trypsin, subtilisin and proteinase K. Bovicin HJ50 was extracted by n-propanol and purified by SP Sepharose Fast Flow, followed by Phenyl Superose and Sephadex G-50. Treatment of Micrococcus flavus NCIB8166 with bovicin HJ50 revealed potassium efflux from inside the cell in a concentration-dependent manner. The molecular mass of bovicin HJ50 was determined to be 3428.3 Da. MS analysis of DTT-treated bovicin HJ50 suggested that bovicin HJ50 contains a disulfide bridge. The structural gene of bovicin HJ50 was cloned by nested PCR based on its N-terminal amino acid sequence. Sequence analysis showed that it encodes a 58 aa prepeptide consisting of an N-terminal leader sequence of 25 aa and a C-terminal propeptide domain of 33 aa. Bovicin HJ50 shows similarity to type All lantibiotics. Chemical modification using an ethanethiol-containing reaction mixture showed that two Thr residues are modified.
Bacteriocins are ribosomally synthesised antimicrobial proteins produced by bacteria that generally kill or inhibit species or strains closely related to the producer. Members of the genus Streptococcus produce a myriad of bacteriocins, most of which are small (10 kDa) streptococcal bacteriocins have also been identified. In this paper, we describe the biochemical and genetic characteristics of the large bacteriocins currently studied in our laboratory, including the identification of stellalysin, a new lytic bacteriocin produced by Streptococcus constellatus subsp. constellatus.
After the discovery of bacteriocin AS-48, a 70-residue cyclic peptide produced by Enterococcus faecalis subsp. liquefaciens, some naturally-occurring cyclic proteins from bacteria have been reported. AS-48 is encoded by the 68-kb pheromone-responsive plasmid pMB2, and the gene cluster involved in production and immunity has been identified and sequenced. This peptide exerts a bactericidal action on sensitive cells (most of the Gram-positive and some Gram-negative bacteria). Its target is the cytoplasmic membrane, in which it opens pores, leading to the dissipation of the proton motive force and cell death, a mechanism similar to that proposed for the action of defensins or, most generally, cationic antibacterial peptides. This fact, together with its remarkable stability and solubility over a wide pH range., suggest that this bacteriocin could be a good candidate as a natural food preservative. The amino acid composition of purified AS-48 shows the absence of modified or dehydrated residues, making it clearly different from lantibiotics. Bacteriocin AS-48 also differs from defensins in that it does not contain cysteines and consequently no disulfide bridges, which makes is high stability even more remarkable. Composition analysis of AS-48 shows a high proportion of basic to acidic amino acids, conferring to this peptide a strong basic character, with an isoelectric point close to 10.5. Determination of the AS-48 structural gene DNA sequence, together with the sequences of AS-48 protease digestion fragments and mass spectrometry determinations, allowed us to determine unambiguously the cyclic structure of the molecule, being the first example of a posttranslational modification in which a cyclic structure arises from a "head-to-tail" linkage. We have solved the three-dimensional structure of AS-48 in solution, and it consists of a globular arrangement of five alpha-helices enclosing a compact hydrophobic core. Interestingly, the head-to-tail peptide link between Trp-70 and Met-1 lies in the middle of alpha-helix 5, which is shown to have a pronounced effect on the stability of the three-dimensional structure. Analysis of structure-function relationship allowed us to propose models to understand the aspects of the molecular function of AS-48. The purpose of this work is to review recent developments in our understanding about the biochemical and biological characteristics and structure of this unusual type of bactcriocin.