Download GENETICS OF BACTERIOCINS BIOSYNTHESIS BY LACTIC ACID

GENETICS OF BACTERIOCINS BIOSYNTHESIS BY
LACTIC ACID BACTERIA
S. Dimov, P. Ivanova, N. Harizanova
Sofia University “St. Kliment Ohridski”, Faculty of Biology, Dept. of Genetics, Sofia,
Bulgaria
Introduction
Chromosomal localization of the bacteriocins operons have been described for the
Cass I, lantibiotics, as well for the Class II
small heat stable peptides. Examples for
chromosomal lantibiotc genes are those
encoding mutacin II and mutacin III produced by Streptococcus mutans (5, 6), as
well the Streptococcus salivarius salivaricin A (7). Chromosomal location of the
class II bacteriocins operons is more widespread. Typical cases are those for enterocins A and B produced by Enterococcus
faecium (8, 9), divercin V41 excreted by
Carnobacterium divergens (10), Lactobacillus johnsonii lactacin F (11), Lactobacillus plantarum plantaricin S (12) and
plantaricin A (13).
Most of the bacteriocins operons are located on plasmids. It has been suggested
that this plasmid location helps the intraand inter-species phylogenetic dissemination of bacteriocins among LAB.
As examples for lantibiotics with plasmid
genetic determinants emerge some well
characterized ones: lacticin 481 (14) and
the two-component lacticin 3147 (15, 16),
both produced by Lactobacillus lactis, and
encoded by genes located on a 70 kb and
63 kb plasmids respectively. Another plasmid determined lantibiotc is the two-component staphylococcin C55 (17) produced
by Staphylococcus aureus C55 harboring a
32 kb plasmid.
Numerous non-lantibiotc bacteriocins
have structural genes located on plasmids.
Typical examples are those for pediocin
PA-1 (18) and pediocin AcH (19) produced
by strains of Pediococcus acidilactici, pos-
Many studies have been focused on bacteriocins produced by lactic acid bacteria
(LAB), especially those produced by many
dairy starter cultures strains because of
their potential industrial application as
natural food preservatives. Bacteriocins are
toxins with protein nature that are ribosomally synthesized by gram-positive bacteria and excreted out of the cell. They can
be divided into 4 major classes: Class I,
lantibiotics (1) (containing posttranslationally modified amino acids such as lanthionine and β-methyllanthionine); Class II
– small unmodified thermo stables usually
positively charged at a neutral pH peptides
(2); Class III – large unmodified thermo
labile peptides (3), and Class IV – complex
bacteriocins which possess a carbohydrate
or lipid component (4). Most of the investigations were focused on the protein nature of bacteriocins, nevertheless in the last
years many data were accumulated regarding the genetic determinants of bacteriocin synthesis as well as its regulation.
This paper is a an attempt for a concise
overview concerning the localization, the
organization and the regulation of the expression of the genes related to bacteriocins
synthesis, procession, export and immunity.
Localization of bacteriocins
genetic determinants
Three types of localizations of the bacteriocins operons have been reported till now
by the authors: on the bacterial chromosome, on plasmids and on transposons
(both plasmids and chromosome carried).
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General & Applied Genetics, Supplement
sessing plasmids with sizes 9.4 kb and 8.9
kb correspondingly; sakicin A determined
by a 60 kb Lactobacillus sakei plasmid
(20); divergicin A from Carnobacterium
divergens (21); the two-component bacteriocin enterocin P synthesized by Enterococcus faecium (22) and many others.
Probably one of the best characterized
bacteriocins is the lantibiotc nisin allowed
in many countries as a food preservative.
Nisin is produced by Lactococcus lactis
and its genetic determinants are located on
the conjugative transposon Tn5276 within
the bacterial chromosome (23). Location on
a mobile genetic element, the transposon
Tn5721, has been also demonstrated for the
lantibiotc lacticin 481 produced also by
Lactococcus lactis but in this case the
transposon is located on a 70 kb plasmid
(24). In the last two cases the bacteriocin
genes are flanked by intact insertion sequences (IS) or inverted repeats (IR). Many
other bacteriocin genes are located in
proximity to defect IS or IR suggesting that
they must be evolved eventually as well
from ancestors on mobile genetic elements.
An unusual genomic organization is described for the non-lantibiotic bacteriocin
carnobacteriocin BM1 produced by Carnobacterium piscicola. While its structural
gene is located on the bacterial chromosome, its expression is dependent on the
presence of a 61 kb plasmid which carries
some of the genes required for the export
and the immunity (25).
are produced by Lactobacillus plantarum
(two two-peptide bacteriocins – plantaricins E/F and J/K, as well as the single
peptide bacteriocin, plantaricin N) (28),
and Enterooccus faecium (one two-peptide
bacteriocin, enterocin L50, and two singlepeptide bacteriocins: enterocin P and enterocin Q) (22).
It is possible that, when more than one
bacteriocin is produced, the peptides can
belong to different classes. For example
Staphylococcus aureus C55 produces the
two-peptide synergetic lantibiotic staphylococcins C55α and C55β, and the non-lantibiotic staphylococin C55γ (17), while
Streptococcus mutans UA140 produces the
lantibiotic mutacin I and the two-peptide
class II bacteriocin mutacin IV (29).
Operon-like organization of
bacteriocin genes
Till now scientific data show that almost all
bacteriocin genetic determinants are clustered in operons or regulons (term proposed by some authors on the basis of the
regulation of the gene expression). This is
not unexpected because in the simplest
case the bacteriocin expression needs at
least two genes: one structural gene and
another one that encodes an immunity protein specific to the produced bacteriocin. In
most cases bacteriocin production needs
also a specific export machinery, and is
subjected to some regulation factors, which
make bacteriocin operons much more complex. Based on the data obtained, there is a
tendency that in general the lantibiotics
operons are more complex than those encoding non-lantibiotics because they need
additional genes encoding enzymes for
posttranslational modifications. With very
few exceptions all bacteriocins are synthesized as prepeptides containing leader sequences.
Some simple bacteriocin operons composed of only two genes are found in the so
called class IIc or sec-dependent bacteriocins. Typical examples are divergicin A
Production of more than one
bacteriocin
Usually LAB produce only one bacteriocin.
But there are many reports for strains producing more than one. Examples for the
secretion of two bacteriocins are the Carnobacterium piscicola carnobacteriocins
BM1 and B2 (25), the Lactococcus lactis
bacteriocins LsbA and LsbB (which can act
synergetically) (26), the Enterococcus faecium enterocins A and B (27) etc. It was
established that three different bacteriocins
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ORF3
SAPK
SAPR
RR
HPK
PRE-PEPTIDE STRUCTURAL
GENE
IMMUNITY PROTEIN
SAPT
SAPE
RIR
LIR
IS1163
ORF4
TRANSPORT PROTEIN
ORF2
ABC-EXPORTER
SAIA
SAPA
ORF1
Fig. 1. Schematic structure of sakicin A operon. The genes related to bacteriocin production are marked with
black colour: sapA – the pre-sakicin A gene, saiA – the gene, encoding the sakicin A immunity protein, sapK –
a histidine protein kinase, sapR – a response regulator, sapT – an ATP binding cassette exporter, sapE – the
transport protein (20).
operon located on 3.4 kb plasmid in Carnobacterium divergens (21), and the Enterococcus faecalis bacteriocin 31 operon
which is located on 57.5 kb conjugative
plasmid (30). These two operons are composed of only two genes: the structural
bacteriocin gene immediately followed
downstream by the gene for the immunity
protein. This simplicity is possible because
the sec-dependent bacteriocins use the general secretory pathway and do not require
dedicated export proteins. There are no
data that the expression of the above two
bacteriocins is under regulation by some
additional factors, that is also confirmed by
cloning and heterologous expression experiments.
More complex is the structure of the
class IIa, pediocin-like bacteriocins. They
contain double-glycine leader peptide and
are exported by dedicated ABC-transporter
(ATP-binding cassette). This subclass of
bacteriocins have their name from some of
the best characterized – pediocins PA-1
and AcH. Pediocin AcH genes are plasmid
located and they are arranged in a gene
cluster of 3500 bp sharing a common promoter and rho-independent stem-loop terminator. The four genes, each with indeBiotechnol. & Biotechnol. Eq. 19/2005/3
pendent ribosome binding sites (rbs), initiation and termination codons and spacer
sequences in between, were designated as
papA, papB, papC and papD (31). PapA is
the structural gene for the pre-pediocin,
while papB encodes the immunity protein.
PapC amd papD form the export machinery, and are required for the membrane
translocation and the removal of the leader
peptide. PapD shares a double-glycine
protease domain with other ABC export
proteins active in transport of bacteriocins.
Sakicin A is another pediocin-like bacteriocin. The sakicin A genes are organized
in two divergent operons (Fig. 1) divided
by an insertion sequence IS1163 (20).
There are six genes participating in the
sakicin A synthesis: sapA –the gene for the
pre-sakicin A, saiA – the gene, encoding
the sakicin A immunity protein, sapK – the
histidine protein kinase, sapR – the response regulator, sapT – the ATP binding
cassette exporter, and sapE – the transport
protein. SapT and sapE are dedicated to the
export of the bacteriocin while sapK and
sapR play a role in the regulation of the
expression via signal transduction mechanisms.
The operons encoding the two-peptide
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A)
LAFA
LAFX
PRE-PEPTIDES
STRUCTURAL GENES
LAFI
IMMUNITY PROTEIN
B)
BRCA
BRCB
PRE-PEPTIDES
STRUCTURAL GENES
BRCI
BRCD
ABC-
IMMUNITY PROTEIN
TRANSLOCATOR
BRCT
RESPONSE
REGULATOR
Fig. 2. Schematic structure of two two-peptide bacteriocin operons. A) Lactacin F operon (11, 32). B) Brochocin-C operon (33).
non-lantibiotic bacteriocins of class IIb, are
in general transcribed in polycistronic
mRNA. Two examples of such operons are
the relatively simple lactacin F operon in
Lactobacillus johnsonii (composed of only
three ORF: lafA and lafX encoding the
prepeptides, and the third, lafI or ORFZ,
encoding the immunity protein (11, 32)),
and the brochocin-C operon in Brochothrix
campestris (33). The second one contains
the structural genes brcA and brcB immediately followed by the gene encoding an
immunity protein brcI. In addition two
other genes – brcD and brcT are located
downstream. BrcD determines an ABCtranslocator while brcT a response regulator. Both operons are compared on Fig. 2.
Similar to brochocin-C operon structure is
found for the divercin V41 operon (10).
Because lantibiotics functionality depends on posttranslational modifications
other than cleavage of the leader sequences, their operons are some of the
most complex among bacteriocins. One of
the best characterized bacteriocin operon is
that encoding the biosynthesis of nisin by
Lactococcus lactis (34). The nisin gene
cluster contains eleven genes and is usually
designed as nisABTCIPRKFEG (Fig. 3).
Of these genes, nisA encodes the nisin A
General & Applied Genetics, Supplement
precursor peptide; nisB and nisC encode
putative enzymes involved in the posttranslational modification reactions; nisT
encodes a putative transport protein of the
ABC translocator family that is probably
involved in the extrusion of the modified
nisin precursor; nisP encodes an extracellular protease involved in precursor processing; nisI encodes a lipoprotein involved
in the producer self-protection against
nisin; and nisFEG encode putative transporter proteins that have also been implied
in immunity. The proteins encoded by nisR
and nisK have shown to be involved in the
regulation of nisin biosynthesis. NisR, a
response regulator, and nisK, is a sensor
histidine protein kinase which belong to the
class of the signal transduction regulatory
systems proteins.
The gene cluster contains two promoters.
The first one is located upstream the nisA
gene, and the second one between the
genes nisP and nisR. It is interesting that an
inverted repeat located between the genes
nisA and nisB could act as a rho-independent terminator suggesting sophisticated
regulation of the expression of the genes
within the nisin cluster.
The following path for nisin biosynthesis
has been proposed. 1) nisA is translated to
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Nisin precursor structural gene
Genes for the enzymes for posttranslational modifications
ABC translocator protein
Transporter proteins implied also in immunity
Extracellular proease involved in the precursor processing
Proteins involved in the regulation of nisin synthesis
Fig. 3. Organization of the gene cluster responsible for nisin synthesis. The genes are marked with different
patterns depending on the function of their protein products (34).
pre-nisin A. 2) Pre-nisin A is transformed
to precursor nisin A by the products of the
genes nisB and nisC. At this stage several
disulfide bridges are made, and as some
amino-acids are transformed to unusual
ones as well. 3) Finally the precursor nisin
A is exported out of the cell by the products
of the genes nisT and nisP at the same time
while the leader peptide is cleaved and the
final product, nisin A is obtained.
Two other well characterized lantibiotic
clusters are those determining lactococcal
lacticin 481 (24) and lacticin 3147 (16).
The first one has analogical structure with
the nisin genes cluster but it contains only
6 genes, all of them transcribed in the same
direction. As the nisin cluster, the lacticin
481 is also framed by IS as a part of the
transposon Tn5721. The lacticin 3147 gene
cluster has a different structure. Although it
contains the same functional groups of
genes there are two major differences: 1)
there are two structural genes for the two
pre-lacticins because lacticin 3147 is a two
peptide lantibiotic, and 2) the genes are
transcribed in two divergent directions.
or by its concurrents for the ecological recesses. However in some cases bacteriocins
production depends on the environmental
conditions (temperature, pH etc) or even it
can be constitutive.
The Enterococcus faecium enterocin B
(9) is an example for constitutive bacteriocin production, while it has been shown
that the expression of enterocins L50A,
L50B and Q produced by another strain of
Enterococcus faecium is affected by environmental changes such as temperature,
ionic strength, and media (22). However
environmental conditions often affect only
the bacteriocin biosynthesis, while the primary regulation is effectuated by appropriate an IF. Such dual mechanism has been proved for sakacin A in Lactobacillus sakei (35).
In many LAB bacteriocin production is
“quorum-sensing” mechanisms controlled.
Quorum-sensing systems control plethora
of different important biological processes
in bacteria, such as natural genetic transformation, virulence, sporulation and many
others. In gram-positive bacteria quorumsensing is predominantly mediated by peptide pheromones or IF. IF are the first component of a three-component signal transduction pathway (Fig. 4).
The induction factor is believed to bind
specifically to the correspondent histidine
protein kinase, and to activate it to phosphorylate the response regulator, which
Regulation of bacteriocins
expression
Expression of bacteriocins genes is usually
subjected to regulation by external induction factors (IF), in most cases small peptides secreted by the producer strain itself
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General & Applied Genetics, Supplement
OUTER SPACE
INNER SPACE
DNA
RR
IF
HPK
PO4
RR
*
TARGET GENES
UNDER ST
CONTROL
CELL MEMBRANE
Fig. 4. Three component signal transduction pathway used by many bacteriocin operons in quorum-sensing
regulation. Abbreviations: IF- induction factor; HPK – histidine kinase; RR and RR* - response regulators.
to be less strictly regulated, and low basal
promoter activity could be detected in uninduced cells. These results show that bacteriocin production in lactobacilli is regulated using a short, strain-specific peptide
pheromone. Similar results have been obtained for another Lactobacillus sakei bacteriocin, sakacin A, where the IF is a
pheromone peptide termed Sap-Ph (35).
But it should also be noticed that in both
sakacin cases the expression is strongly
influenced by the temperature conditions.
Typical case in which the bacteriocin
molecule itself plays a role of an IF is nisin
(34). In this case the mature nisin molecule
interacts with a sensor histine protein
kinase (HPK), produced by the gene nisK,
and disposed on the cell membrane. The
HPK phosphorylates the response regulator
(RR), product of the gene nisR, followed
by binding to the imperfect direct repeats
found in the promoter regions. This event
leads to a transcription activation of the
nisin gene cluster, and finally to the secretion of more nisin molecules, thus closing
the cycle of autoactivation.
than stimulates transcription of the target
genes, most probably by binding to specific
imperfect direct repeats found in many
bacteriocin genes clusters. IF for a given
bacteriocin operon can be produced either
by the bacteriocin producer strain, or by
other strains belonging to the same or other
species or genera.
When the inducer peptide is produced by
the bacteriocin producer itself there is an
autoregulation of bacteriocin biosynthesis.
The IF can be a dedicated peptide encoded
by a respective gene (exported by specific
ABC-translocator or by the general secretory
pathway), or the bacteriocin molecule itself.
Production of the bacteriocin sakacin P
by Lactobacillus sakei LTH673 is dependent on a secreted 19-residue peptide
pheromone (IP-673) (36). The gene encoding IP-673, SppIP, is co-transcribed
with the genes encoding the histidine protein kinase (sppK) and the response regulator (sppR). IP-673 induces the transcription of its own gene and of what are often
considered to be of all genes necessary for
the bacteriocin production and immunity.
Studies with a reporter gene showed that
the promoter in front of the sakacin P
structural gene (sppA) is strictly regulated.
The promoter in front of sppIP turned out
General & Applied Genetics, Supplement
Conclusions
Bacteriocins represent enormous interest
for food industry because they are produced by many dairy starter culture strains,
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and because of their capability to repress
the development of pathogenic microflora
in dairy products. Their potential application as natural food preservatives depends
on the capacity of expression of bacteriocin
genetic determinants by genetically modified heterologous host strains at industrial
level. This task will be unimaginable without a deep understanding of the bacteriocins genetics. In the last decades many
scientific results were obtained which inevitably led to the industrial production and
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preservatives against food spoilage. The
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