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REVIEW ARTICLE
Cannibalism: a social behavior in sporulating Bacillus subtilis
José Eduardo González-Pastor
Department of Molecular Evolution, Centro de Astrobiologı́a (CSIC-INTA), Madrid, Spain
Correspondence: José Eduardo GonzálezPastor, Department of Molecular Evolution,
Centro de Astrobiologı́a (CSIC-INTA),
Carretera de Ajalvir km 4, Torrejón de Ardoz
28850, Madrid, Spain.
Tel.: 134 91 520 6470;
fax: 134 91 520 1074;
e-mail: [email protected]
Received 8 March 2010; revised 15 August
2010; accepted 10 September 2010.
Final version published online 19 October 2010.
DOI:10.1111/j.1574-6976.2010.00253.x
Abstract
A social behavior named cannibalism has been described during the early stages of
sporulation of the Gram-positive Bacillus subtilis. This phenomenon is based on
the heterogeneity of sporulating populations, constituted by at least two cell types:
(1) sporulating cells, in which the master regulator of sporulation Spo0A is active,
and (2) nonsporulating cells, in which Spo0A is inactive. Sporulating cells produce
two toxins that act cooperatively to kill the nonsporulating sister cells. The
nutrients released by the dead cells into the starved medium are used for growth
by the sporulating cells that are not yet fully committed to sporulate, and as a
result, sporulation is arrested. This review outlines the molecular mechanisms of
the killing and immunity to the toxins, the regulation of their production and
other examples of killing of siblings in microorganisms. The biological significance
of this behavior is discussed.
Editor: Bernardo González
MICROBIOLOGY REVIEWS
Keywords
cannibalism; fratricide; sporulation; biofilms;
multicellularity; bacteriocins.
Introduction
Microorganisms produce ‘chemical weapons’ to protect
themselves from other microorganisms that could compete
for the same niche and resources. These compounds are
designed to inhibit growth or to kill a broad array of
microbial species or just closely related strains of the same
species. The production of antimicrobial compounds also
involves the development of an immunity system to prevent
the killing of the producer cells. This ‘chemical warfare’ is
coordinated by the whole population to attack those microorganisms sharing a common niche; thus, all members of
the population are simultaneously producers and also
immune to the toxins. Unexpectedly, it has been reported
that some members of a genetically identical population can
kill their siblings using antimicrobials. In the case of Bacillus
subtilis, cells at the onset of sporulation secrete extracellular
killing factors that lyse sibling nonsporulating cells that have
not developed immunity to these toxins. This killing releases
nutrients from the dead cells into the starved medium that the
surviving sporulating cells can feed on, and thus this behavior
was termed cannibalism (Fig. 1a) (González-Pastor et al.,
2003). In Streptococcus pneumonia, a fratricide behavior was
FEMS Microbiol Rev 35 (2011) 415–424
described in which cells that are competent for natural
genetic transformation express proteinaceous toxins that
will lyse noncompetent siblings (Steinmoen et al., 2002,
2003; Guiral et al., 2005; Håvarstein et al., 2006). A common
feature of this phenomenon in these microorganisms is that
it is based on a differentiation process: sporulation in the
case of B. subtilis and competence in S. pneumoniae.
Cannibalism and fratricide were compared and reviewed by
Claverys & Håvarstein (2007).
In B. subtilis, cannibalism is associated with sporulation,
but not directly with competence, because this phenomenon
is not affected by mutations in the main regulator of
competence, comA (O. Zafra & J.E. González-Pastor, unpublished data; Nakano et al., 1991a, b). On the other hand,
it cannot be excluded that the DNA released might be
integrated by competent cells. Spore formation is initiated
by nutrient limitation and involves the asymmetric division
of the developing cell to produce the forespore and the
mother cell, which follow different pathways of differentiation (Fig. 1b) (Piggot & Coote, 1976; Sonenshein, 2000;
Piggot & Losick, 2002; Errington, 2003). The mother cell
engulfs the forespore, and contributes to the transformation
of the forespore to spore, but ultimately the mother cell
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416
J.E. González-Pastor
Fig. 1. (a) Model for cannibalistic behavior.
Bacillus subtilis cells enter into sporulation under
nutrient limitation conditions. Spo0A, the master
regulator of sporulation, becomes active only in a
part of the population. The Spo0A-active cells
produce and are immune to the Skf and Sdp
toxins. The Spo0A-inactive cells are sensitive to
the toxins because (1) the ABC transporter and
other putative immunity genes involved in Skf
resistance are not transcribed and (2) AbrB is
expressed in Spo0A-inactive cells (abrB gene is
repressed by Spo0A), and repress transcription of
the operon sdpRI conferring immunity to the Sdp
toxin. Therefore, sporulating cells kill the
nonsporulating siblings. The nutrients from the
dead cells are released into the starved medium,
and those sporulating cells that are not yet
committed to sporulate feed on these nutrients
and resume growth. As a result, sporulation is
arrested. (b) The sporulation cycle of spore
formation. The key stages of the cycle are shown.
undergoes lysis (Fig. 1b). Cells are not fully committed to
sporulate until they form an asymmetric polar septum.
Before that, they can resume growth if provided with
nutrients. However, cells that have passed the asymmetric
division stage will complete spore formation even if nutrients are supplied (Parker et al., 1996). Sporulation is a very
complex developmental process that requires plenty of
energy and needs several hours to be completed. Hence, the
commitment stage reflects the need for a checkpoint in this
elaborated process, to make it reversible in case nutrients are
again available to the population. The killing of nonsporulating cells releases nutrients from the dead cells into the
starved medium so that the sporulation process is arrested
in those cells not yet committed to sporulate, which can
grow and divide again (González-Pastor et al., 2003) (Fig.
1a). Here, we focus on different aspects of the cannibalistic
behavior, such as molecular mechanisms of killing and
immunity, regulation, biological significance of cannibalism
and other examples of fratricide in microorganisms.
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Molecular mechanisms of cannibalism
The killing of nonsporulating cells is controlled by two
independent gene clusters (Fig. 2): skf for sporulation killing
factor, and sdp for sporulation delaying protein. Both
clusters were previously identified as positively regulated by
Spo0A, the master regulator of sporulation (Fawcett et al.,
2000), and in fact, they are highly expressed at the onset of
sporulation. Interestingly, mutants in each cluster exhibit an
accelerated sporulation phenotype when growing on solid
medium. Previous studies on sporulation were focused on
the characterization of defective mutants, but this was the
first time that mutants with faster sporulation were studied.
The relevance of this phenotype will be discussed below.
The skf operon contains eight genes (skfA-H, Fig. 2)
involved in the production and release of a killing factor,
and in the immunity to it, and it is similar to operons
involved in the synthesis of bacteriocins (ribosomally
synthesized antimicrobial compounds). skfA, the first gene
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Cannibalism during sporulation
of the operon, encodes a 55-amino-acid peptide, which
could constitute the precursor of the killing factor. skfB
encodes a protein similar to AlbA, a B. subtilis protein
involved in the post-translational modification of subtilosin,
a ribosomally synthesized antimicrobial peptide (Zheng
et al., 1999). The product of skfD contains a domain
conserved in the type II CAAX family of prenyl endopeptidases (Pei & Grishin, 2001), and it could be involved in SkfA
maturation or immunity by cleaving the mature killing
factor. Finally, the products encoded by skfE and skfF
resemble the two components of an ATP-binding cassette
transport complex (ABC transporter), the nucleotide-binding protein and the permease, respectively. These transporters are typically found in bacteriocin biosynthesis operons,
and they are responsible for the secretion of the bacteriocin
and also for conferring resistance to it.
Bacteriocins typically contain a leader peptide identified
by a consensus sequence and a GG-motif (Michiels et al.,
2001). SkfA contains the double-glycine sequence but lacks
the consensus sequence of the leader peptide, and in addition, the putative ABC transporter, SkfEF, which seems to be
required for the transport of the killing factor, lacks the
proteolytic domain required for the processing of the leader
peptide (Michiels et al., 2001). Thus, SkfA maturation could
be carried out by the the putative endopeptidase activity of
SkfE or by the unknown products of skfC, skfG or skfH.
The killing factor produced by the skf operon has not yet
been purified, but a set of experiments have proven its
activity (González-Pastor et al., 2003). (1) Wild-type cells
kill skf mutant cells (deletion of different skf genes and the
deletion of the whole operon). If skf and wild-type cells are
equally mixed in liquid sporulation medium, the ratio of skf
to wild-type cells remain constant during growth, but
decreases drastically after the onset of sporulation. Thus,
the skf operon is involved in the production of a killing
factor during sporulation, and also encodes resistance to it.
In fact, when the skfEF genes encoding the ABC transporter
are overexpressed in an skf mutant, these cells become
resistant to the killing factor. (2) Cells modified to express
the skf operon during growth, under control of an isopropyl-b-D-thiogalactopyranoside (IPTG)-inducible promoter,
produce a halo of growth inhibition when spotted on a lawn
of wild-type or skf mutant, only in the presence of the IPTG
inducer. (3) Viability during sporulation is not affected in a
culture of an skf mutant. Approximately two-thirds of the
cells in a sporulating population of a wild-type strain are
killed as a result of the killing factor encoded by the skf
operon.
The second cluster of genes also involved in the killing is
constituted by two convergent operons: sdpABC and sdpRI
(Fig. 2). The sdpABC operon is responsible for the production and secretion of a 63-amino-acid protein that is derived
from the C-terminal portion of the product of sdpC.
Initially, the SdpC peptide was described as a signal involved
in delaying sporulation by inducing the expression of the
adjacent genes sdpRI (González-Pastor et al., 2003). Recently, SdpC has been shown to be a toxin, and the products
of sdpRI confer immunity to the toxin (Ellermeier et al.,
2006). The transcription of the sdpRI genes is strongly
affected by a mutation in sdpC as revealed by DNA microarrays, but transcription is restored if mutant cells are grown
close to the wild-type cells (González-Pastor et al., 2003).
The SdpC protein can be purified from conditioned medium from sporulating wild-type cultures, eluted from
reverse-phase chromatography, and SdpC corresponds to a
predominant and very intense band of approximately 5 kDa
(González-Pastor et al., 2003). The purified SdpC protein
stimulates transcription of the sdpRI genes. SdpI, which is
predicted to be an integral membrane protein, confers
immunity to SdpC (Ellermeier et al., 2006). Competition
experiments with mixtures of wild-type and sdp mutant cells
support the role of SdpC as a toxin. Wild-type cells kill
sdpABC sdpRI mutant cells but not sdpABC mutant cells,
and the killing of sdpABC sdpRI is overcome when sdpI is
overexpressed. The SdpR protein exhibits similarity to the
ArsR family of regulators, and it was previously identified in
a screening for genes that inhibit the expression of sigW, a
gene involved in detoxification and resistance to antibiotics
(Turner & Helmann, 2000; Cao et al., 2002). SdpR directly
regulates the sdpRI operon by binding to its promoter
region, and acts as an autorepressor inhibiting transcription
of these genes. As described previously, transcription of the
sdpRI operon is abolished in an sdpC mutant, but it is
restored by an additional mutation in sdpR, which proves its
autorepressor activity. The SdpI immunity protein is also
considered a signal transduction protein directly involved in
the response of sdpRI to SdpC signalling (Ellermeier et al.,
2006). An sdpI null mutant does not exhibit SdpC-stimulated expression of the sdpRI operon. Furthermore, sdpI
mutants blocked in an activated state for sdpRI transcription
Fig. 2. Gene organization for the skfABCDEFGH
and the sdpABC sdpRI operons. The hairpin
symbols represent transcriptional terminators.
FEMS Microbiol Rev 35 (2011) 415–424
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418
in the absence of the SdpC signal and sdpI mutants unable to
induce a transcriptional response in the presence of SdpC
were obtained, and both types of mutants retain the capacity
to confer immunity.
How do SdpC and SdpI prevent repression by SdpR? The
repressor activity of SdpR is regulated by membrane sequestration in an SdpC–SdpI-dependent manner. SdpR localization was studied using a green fluorescent protein (GFP)
functional fusion. In wild-type cells, SdpR-GFP localizes to
the cytoplasmic membrane, but in sdpC or spdI mutant cells,
the fusion proteins are homogeneously distributed throughout the cytoplasm. Therefore, it was proposed that SdpR can
be sequestered at the membrane by the SdpI–SdpC complex,
and it would be inactive (Fig. 3) (Ellermeier et al., 2006).
The cannibalism in sporulating cultures is a consequence
of the population heterogeneity regarding Spo0A activation.
It has been reported that isogenic bacteria grown under
identical conditions do not display the same pattern of gene
expression, which is termed bistability. In the absence of
genetic modifications, there is a reversible switching between different states (Dubnau & Losick, 2006). In a culture
of sporulating B. subtilis, less than half of the population
contains activated Spo0A (Chung et al., 1994; GonzálezPastor et al., 2003). The auto-stimulatory loops controlling
Spo0A activity of spo0A are involved in the generation of a
bistable response (Veening et al., 2005). Therefore, the
killing factors and the resistance to it will be produced by
Spo0A-active cells, but not by Spo0A-inactive cells, which
will be killed.
J.E. González-Pastor
Immunity, how to be protected from the
killing factors?
Why are the sporulating cells immune to the toxins that they
produce? Immunity towards the Skf killing factor is not
completely characterized. As mentioned previously, SkfE
and SkfF could constitute an ABC transporter, and experimental evidence shows that it is involved in the pumping of
this killing factor, conferring resistance to it. A similar
involvement of an ABC transporter in the self-protection
mechanism has been described for different antimicrobial
peptides, such as subtilosin (AlbC and AlbD) (Zheng et al.,
2000) and subtilisin (SpaF and SpaG) (Klein & Entian,
1994) from B. subtilis, and microcin B17 (McbE and McbF)
from Escherichia coli (Garrido et al., 1988). In addition to
the genes encoding the ABC transporters, other genes
involved in immunity have been described in these systems.
Therefore, additional immunity mechanisms could be provided by other genes in the skf operon, for instance the skfD,
whose product contains a domain that shows homology to
the type II CAAX prenyl endopeptidases. This domain is
also present in the putative bacteriocin immunity proteins
PlnP, PlnI, PlnT and PlnU from Lactobacillus plantarum and
BlpY from S. pneumoniae (Pei & Grishin, 2001; Claverys &
Håvarstein, 2007), and they could inactivate bacteriocins by
proteolytic cleavage.
The sporulating cells are also protected from SdpC,
because the immunity protein SdpI is induced in the
presence of SdpC. However, why are nonsporulating cells
killed by the SdpC toxin? SdpC as an extracellular signal
could also activate transcription of the immunity gene sdpI
in the nonsporulating cells. Spo0A regulator represses the
synthesis of the unstable AbrB regulator, which is a repressor; thus, AbrB is expressed and it blocks transcription of the
sdpRI operon in nonsporulating cells. It was shown that the
sdpRI genes are not transcribed in an spo0A mutant, but they
are transcribed at a high level in an abrB and also in an spo0A
abrB double mutant (Ellermeier et al., 2006). Therefore,
only sporulating cells would be immune to the SdpC toxin,
because activation of the transcription of sdpI requires
activated Spo0A (which is not the case in nonsporulating
cells), in order to relieve repression through AbrB.
Why do skf and sdp mutants sporulate
faster?
Fig. 3. Model of how SdpC induces expression of the sdpRI immunity
operon. The signal SdpC (C) interacts with the immunity protein SdpI to
induce a conformational change that allows SdpI to bind the repressor
SdpR (R), which is sequestered at the membrane. This mechanism
prevents SdpR from binding at the promoter of the sdpRI operon,
thereby relieving repression. In the absence of SdpC, SdpR is in the
cytoplasm, and not sequestered at the membrane, and it binds at the
sdpRI promoter and represses its transcription.
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The accelerated sporulation phenotype of the skf and sdp
mutants reveals that the sporulation process in B. subtilis
wild-type cells is delayed. The killing factors produced by
these operons at the first signals of starvation lyse nonsporulating cells, which are sensitive to killing. Then, nutrients are
released into the medium, and those cells in which Spo0A is
active, but which have not yet committed to sporulation, can
FEMS Microbiol Rev 35 (2011) 415–424
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Cannibalism during sporulation
use those nutrients and resume growth during stationary
phase. As a result, spore development is arrested until the new
nutrients are exhausted. Again, starvation will divide the
remaining cells into Spo0A-active and -inactive populations,
and new killing episodes will take place until most of the
population is transformed into spores. These cycles of killing
events during stationary phase are responsible for a significant delay of sporulation in wild-type cells. In the absence of
one or two of the killing factors, Spo0A-inactive cells are not
killed, no more nutrients are released into the exhausted
medium and therefore spore formation is not arrested. The
biological significance of the delay in sporulation produced
by the cannibalism is discussed below.
Killing factor production is induced by
low levels of active Spo0A
Spo0A is known as the master regulator of the initiation of
sporulation, and is also important in the intermediate stages
of sporulation, precisely in the mother cell compartment
where it accumulates (Fujita & Losick, 2003). Activated
Spo0A directly controls 121 genes organized in 30 single
genes and 24 operons or clusters (Fawcett et al., 2000; Molle
et al., 2003). However, not all these genes respond equally to
Spo0A; some of them are activated at low concentrations of
Spo0A, and others only at high concentrations (Chung et al.,
1994). More recently, transcriptional-profiling experiments
using DNA microarrays were applied to identify the Spo0Adependent genes distributed in each category (Fujita et al.,
2005). It was observed that genes requiring a high dose of
Spo0A to be activated have a low binding affinity for this
regulator. Genes directly involved in sporulation, such as
spoIIA, spoIIE and spoIIG, are included in this category. On
the other hand, genes activated by a low dose of Spo0A
either have a high-affinity binding site or are indirectly
regulated by Spo0A, which relieves the repression by the
AbrB regulator. The operons involved in cannibalism,
skf and sdpABC, fall into this category. The promoter of the
skf operon has a high-affinity binding site for Spo0A, and
the sdpABC operon is repressed by AbrB, and therefore, is
indirectly activated at a low concentration of Spo0A through
repression of abrB (Fujita et al., 2005). Moreover, AbrB
directly represses transcription of the skf operon; thus, skf is
activated by two routes: directly by Spo0A and indirectly by
relieving AbrB-mediated repression. What is the biological
significance of having differential responses to high and low
doses of Spo0A? At an early stage of sporulation, activated
Spo0A is produced at a low level in the cells, and this turns
on genes involved in auxiliary roles in development, like for
instance cannibalism and formation of multicellular aerial
structures (Fujita et al., 2005), in which sporulation will take
place (Fig. 4). The building of these multicellular structures
can be seen as a prelude to spore formation. Then, if
FEMS Microbiol Rev 35 (2011) 415–424
Fig. 4. Different levels of active Spo0A (Spo0A phosphorylated, Spo0AP) control cannibalism, formation of multicellular structures and sporulation. The operons involved in the killing of the nonsporulating sibling
cells, skf and sdp, are regulated by a low dose of Spo0A-P. The skf operon
is repressed by AbrB. sdpABC and sdpRI are also repressed by AbrB, and
thus indirectly activated by a low dose of Spo0A-P through repression of
abrB. The formation of multicellular structures is also induced at a low
dose of Spo0A-P. In contrast, sporulation is triggered only at a higher
concentration of active Spo0A.
conditions still promote sporulation, there is a progressive
increase in the intracellular concentration of activated
Spo0A, and the genes that play a direct role in spore
formation are turned on (Fig. 4).
What is the biological significance of
cannibalism?
A B. subtilis population could be at risk if most of the
members engage in sporulation at once. Spore formation
consumes plenty of energy and time to be completed, and it
is reversible only up to a certain point, as described
previously (Parker et al., 1996). For instance, sporulating
cells and spores could not resume growth as efficiently as
vegetative cells or even resting cells if nutrients were
available again in the medium. In natural environments,
B. subtilis communities are surrounded by other microorganisms, many of them nonsporulating, and B. subtilis cells
committed to sporulate or spores could be at disadvantage
relative to them. Cannibalism helps to sustain a mixed
population during the stationary phase with a small percentage of spores and a high percentage of growing cells for a
longer period of time, which might be beneficial to the
community. In the absence of cannibalism, as in skf or sdp
mutants, the whole population initiates sporulation
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420
simultaneously, and it could face problems to recover easily
when nutrients are again available. Obviously, the toxins
produced by the sporulating cells could also kill other
microorganisms in the environment, which could also
provide the nutrients to delay sporulation. In fact, Skf is
active against Xanthomonas oryzae, a Gram-positive plant
pathogen (Lin et al., 2001), which supports the fact that Skf
could be a broad-spectrum bacteriocin, and could kill other
soil-inhabiting bacteria. The fact that these toxins could also
kill different microbial species does not rule out that they
can also be used more specifically against B. subtilis cells
from the same population.
It could be interesting to test whether the presence of
different microorganisms inhibits the killing of B. subtilis
siblings. Nandy et al. (2007) have demonstrated that sporulating bacteria prefer predation of other microorganisms to
cannibalism in mixed cultures. Cells from stationary cultures
of E. coli and B. subtilis were mixed in a phosphate buffer
solution, devoid of any nutrients. In this medium,
B. subtilis was not able to sporulate, but the viability shows
an oscillatory behavior, which can be explained by the killing
and the growth of sister cells through cannibalism. In fact, the
viability of an skf mutant does not exhibit this oscillatory
behavior. In the mixing experiment, viability of E. coli is
drastically reduced (eight log fold change) during the first
10 h, and no oscillation is observed in the viability of
B. subtilis. However, after 10 h, viability of B. subtilis starts an
oscillatory behavior, which indicates the cannibalistic behavior. It was shown that the predation of E. coli cells was not
due to the killing factor Skf, but due to other bacteriocins
controlled by Spo0A, because the Spo0A mutant cannot kill E.
coli cells in the mixing experiments. In any case, these
experiments did not show that the presence of other microorganisms inhibits the killing of sister cells. What happens is
that E. coli cells are killed by other bacteriocins produced at the
onset of sporulation, and the nutrients released delay the
sporulation process. When the medium is exhausted, sporulation can be resumed again in B. subtilis, initiating cannibalistic
behavior. It would be interesting to show that in the presence
of other microorganisms, the killing factors preferentially act
on these, but not on the nonsporulating sister cells.
It has been proposed that cannibalism could be required to
eradicate competitors, nonsporulating B. subtilis cells or other
species that could use the nutrients needed to complete the
complex sporulation process (Claverys & Håvarstein, 2007).
Spore formation is induced while there are still enough
nutrients available to complete the process. However, if other
cells are growing in the medium, the nutrients could be
exhausted before the cells become spores. Against this view, it
was observed that in cultures of an skf mutant, in which
sporulating and nonsporulating cells were present, sporulation
takes place perfectly, and spores are morphologically identical
to wild-type spores and they are heat resistant (González2010 Federation of European Microbiological Societies
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J.E. González-Pastor
Pastor et al., 2003). In addition, it should be noticed that in
cultures of wild-type cells, the availability of nutrients after the
killing of nonsporulating cells induces the growth of those
sporulating cells that are not yet committed. This growth
happens when committed cells are completing the sporulation
process, but the use of nutrients by the growing cells appears
not to affect the completion of sporulation.
Recently, it has been proposed that cannibalism plays a
stimulatory role in the development of multicellular communities or biofilms in B. subtilis (López et al., 2009b). Undomesticated but not laboratory strains of this bacterium
(Branda et al., 2001) form aerial multicellular structures in
which sporulation takes place. The master regulator of
sporulation, Spo0A, indirectly regulates biofilm formation
through AbrB (Branda et al., 2001; Hamon & Lazazzera,
2001) and SinI/SinR (Kearns et al., 2005; Chu et al., 2006)
(Fig. 5). SinR is a DNA-binding protein that represses the
expression of the genes involved in the production of the
extracellular matrix: epsA-O, producing an exopolysaccharide
(Branda et al., 2001), and yqxM-sipW-tasA, producing matrix
protein TasA (Branda et al., 2006). SinI is an antagonist of
SinR with which it forms a complex (Gaur et al., 1991; Bai
et al., 1993; Lewis et al., 1998). In Spo0A-active cells, SinI is
expressed and counteracts the SinR repressor, thus relieving
the repression of the genes required for matrix formation.
Vlamakis et al. (2008) described that a specialized subpopulation of cells differentiates within the multicellular aerial
structures, and produces the exopolysaccharide and TasA
components of the extracellular matrix. More recently, it has
been shown that the same population of cells producing this
matrix also produces the cannibalism toxins, Skf and Sdp
(López et al., 2009b). The killing of the sibling nonsporulating
cells releases nutrients, and then, the growth of matrixproducing cells is promoted, matrix production is increased
and sporulation is temporarily delayed. A ‘hypercannibal’
mutant was used to demonstrate that cannibalism contributes to biofilm development. The hypercannibal mutant
contains mutations in (1) abh, which encodes an AbrB
homolog (Yao & Strauch, 2005; Bobay et al., 2006) and is a
repressor of both skf and sdp operons (López et al., 2009b),
and (2) dlt (dltABCDE), an operon encoding the machinery
responsible for the D-alanine esterification of cell wall teichoic
acids (Perego et al., 1995). The dlt mutant is more sensitive to
the action of cannibalism toxins. This so-called ‘hypercannibal’ mutant overexpresses the toxins, and is more sensitive to
them. The morphology of the hypercannibal mutant colonies
is more wrinkled compared with the wild type, and this was
related to an increase in the production of extracellular
matrix, which supports the connection between cannibalism
and matrix production (López et al., 2009b). However, an skf
or an sdp mutant seems not to be affected in the formation of
the multicellular structures (J.E. González-Pastor, unpublished data).
FEMS Microbiol Rev 35 (2011) 415–424
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Cannibalism during sporulation
Fig. 5. Model for biofilm formation in Bacillus
subtilis. Spo0A is the master regulator for the
formation of aerial structures, indirectly through
SinI/SinR, and AbrB controls the expression of the
eps, yqxM and tasA genes required for the
production of the extracellular matrix of the
biofilm. Spo0A is also involved in the cannibalism
behavior controlling the expression of the genes
involved in the toxin production, directly on the
skf operon, and indirectly through AbrB on the
sdpABC operon (proteins that are not expressed
and processes that are turned OFF are indicated
in gray). In Spo0A-active cells, (1) SinI is expressed
and counteracts the SinR repressor, thus relieving
the repression of eps, yqxM and tasA, and (2)
AbrB is repressed; thus, the repression of tasA is
also relieved. In Spo0A-inactive cells, SinR
represses the formation of the matrix and
indirectly favors cell division and motility. The
aerial structures are formed by cells entering
sporulation, and heterogeneity in Spo0A
activation has been reported to exist in these
populations. Spo0A-active and Spo0A-inactive
cells, and also other types of cells such as the
ones expressing competence, are part of these
communities, and they are spatially organized in
the aerial structures (Branda et al., 2001;
Vlamakis et al., 2008). Surfactin has been
recently described as a quorum-sensing molecule
that activates KinC, a membrane histidine kinase
involved in the process of Spo0A activation by
phosphorylation.
Another interesting finding was that the lipopeptide
surfactin triggers both cannibalism and matrix production
in the same subpopulation of cells (López et al., 2009b).
Surfactin has been recently identified as a quorum-sensing
molecule, which activates the membrane histidine kinase
KinC with the consequent phosphorylation of Spo0A
(López et al., 2009a). KinC senses leakage of potassium
caused by the action of surfactin; in fact, other antimicrobial
compounds with similar physiological effects, such as nystatin, amphotericin, valinomycin and gramicidin, can also
activate KinC. This suggested that other soil bacteria producing compounds with effects similar to surfactin could also
trigger cannibalism and the development of multicellular
communities (López et al., 2009b).
Other examples of fratricide in
microorganisms
A case of fratricide, named allolysis, has also been wellcharacterized in the Gram-positive pathogenic bacterium S.
pneumoniae (Guiral et al., 2005; Håvarstein et al., 2006).
Cells that are competent for natural genetic transformation
FEMS Microbiol Rev 35 (2011) 415–424
lyse noncompetent cells, and virulence factors are released.
In contrast to sporulation, nutritional stress does not trigger
the development of the competence state in S. pneumoniae,
which has been proposed to constitute a general stress
response (Claverys et al., 2006). Under laboratory conditions, competence is induced in rich medium during early
logarithmic growth phase, and develops in response to
different environmental signals (Claverys & Håvarstein,
2002), such as changes in pH or the presence of antibiotics
(Claverys et al., 2006; Prudhomme et al., 2006). Competence
is induced by the competence-stimulating peptide (CSP)
(Claverys & Håvarstein, 2002; Claverys et al., 2006), which is
secreted by ComAB. Interaction of CSP with its receptor,
ComD, a membrane-bound histidine kinase, leads to ComD
autophosphorylation, followed by transfer of the phosphoryl group to the ComE response regulator. ComEP directly
activates the expression of approximately 20 early competence genes (Dagkessamanskaia et al., 2004; Peterson et al.,
2004). One of these genes is comX, encoding an alternative
sigma factor that controls the expression of approximately
60 late competence genes (Luo & Morrison, 2003; Dagkessamanskaia et al., 2004; Peterson et al., 2004). Four of them
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c
422
are required for fratricide (Guiral et al., 2005): CbpD is a
novel murein hydrolase (Eldholm et al., 2010), LytA is the
major pneumococcal autolysin and the other two, CibA and
CibB (CibAB), presumably constitute a two-peptide bacteriocin. An additional autolytic enzyme, not induced in
response to CSP, is also involved in fratricide, LytC (Garcı́a
et al., 1999; Guiral et al., 2005). These fratricide killing
factors are expressed in parallel with the proteins involved in
DNA processing, uptake and recombination. Two predicted
integral membrane proteins protect competent cells from
lysis. One is the product of cibC, cotranscribed with cibAB,
which could confer immunity to the bacteriocin. Competent cells with a mutation in cibC are sensitive to lysis
(Guiral et al., 2005). The second immunity factor is encoded
by an early competence gene, ComM, which provides
immunity to the combined lytic action of CbpD and LytALytC (Håvarstein et al., 2006). The precise mechanisms by
which CibB and ComM confer immunity remain unknown.
What is the biological relevance of fratricide in S.
pneumoniae? Taking into account the fact that competence
is not induced by nutrient limitation, the release of transforming DNA, pneumolysin and other relevant factors
could facilitate the invasion of S. pnenumonie into its host
(Guiral et al., 2005). In addition, it has been suggested that
fratricide may provide genetically diverse DNA to generate
diversity by genetic transformation (Claverys & Håvarstein,
2007), and experimental evidence strongly supporting a role
for fratricide in lateral gene transfer has been published
(Johnsborg et al., 2008).
The phenomena of cannibalism and fratricide share some
common features: (1) Both are based on cell differentiation
in a bacterial population, sporulation in B. subtilis and
competence in S. pneumoniae, and require a mixed population of cells. In contrast to cannibalism, fratricide is not
based on a natural heterogeneity of the population. Under
laboratory conditions, using the most-studied laboratory
strains, there is no evidence of bistability, and the level of
competence of the cultures approaches 100%. The fratricide
behavior was discovered by artificially mixing populations
of competent and noncompetent cells. Futures studies on
natural isolates and the use of different laboratory conditions could reveal a natural heterogeneity in S. pneumoniae
populations. (2) Both phenomena involve the synthesis of
two sets of killing factors and the corresponding immunity
machinery, which seem unrelated. A more detailed comparison of cannibalism and fratricide is described in Claverys &
Håvarstein (2007).
Interestingly, in another sporulating bacterial genus,
Myxobacteria, it has been described that autolysis occurs in
stationary-phase cultures, and this seems to happen during
fruiting body formation. Approximately 80% of the initial
vegetative population undergoes lysis (Wireman & Dworkin, 1977). This massive lysis is proposed to have a func2010 Federation of European Microbiological Societies
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c
J.E. González-Pastor
tional role in the formation of myxospores in the fruiting
bodies (Wireman & Dworkin, 1977). The toxins involved in
this killing are called autocides, and they are expressed
under conditions in which fruiting body formation is
induced (Varon et al., 1984). Interestingly, these autocides
are active only against the producing and closely related
strains, but not against other microorganisms. Therefore, in
Myxobacteria, the killing has been suggested to be a mechanism of programmed cell death (Nariya & Inouye, 2008).
Concluding remarks
The work summarized in this review demonstrates the
relevance of the study of the behavior of microorganisms
from a multicellular and developmental perspective, rather
than from an autonomous cell perspective (Shapiro, 1998;
Aguilar et al., 2007). Cannibalism, sporulation and formation of multicellular aerial structures are connected processes, controlled by the same regulator in B. subtilis, Spo0A.
This regulator has been extensively studied as the master
regulator of sporulation at the unicellular level. However,
research on cannibalism and the formation of multicellular
structures places the role of Spo0A into a much broader
perspective. Under nutritional stress, this regulator is activated only in a part of the population; first, at low
concentrations, it is involved in the formation of aerial
structures and the killing of nonsporulating cells. Then, if
conditions still promote sporulation, active Spo0A reaches a
higher concentration and directly activates spore formation.
These complex properties of Spo0A resemble those of
eukaryotic developmental regulators. In this context, cannibalism behavior exerts an additional control on the sporulation process by the bacterial community: (1) it is involved in
the delay of sporulation and (2) it seems to play a role in the
development of the aerial multicellular structures in which
spores are formed.
Population heterogeneity to develop complex behaviors is
thus of high relevance. Cannibalism in B. subtilis is entirely
based on the Spo0A bistable switch, which is responsible for
the generation of cell types with different patterns of gene
expression. Otherwise, the other features of this behavior,
such as the killing factors and the immunity mechanisms,
are not especially different from others described previously.
The ability of B. subtilis to generate different cell types is the
driving force for the different examples of social behavior in
this bacterium, like for instance, the formation of aerial
structures, which requires the coordination of different cell
types (reviewed in González-Pastor, 2006).
Future research might be oriented to explore other social
behaviors based on population heterogeneity in B. subtilis
and in other bacteria exhibiting different developmental
pathways. Not all the genes controlled by the main regulators of a developmental pathway are directly involved in the
FEMS Microbiol Rev 35 (2011) 415–424
423
Cannibalism during sporulation
generation of a different cell type; some of them might be
involved in auxiliary functions and novel social behaviors
that are related indirectly. This is the case for the genes
encoding the killing factors or toxins in B. subtilis sporulating cells or in S. pneumoniae competent cells.
Acknowledgements
The author is very grateful to Olga Zafra for her helpful
comments on the manuscript, and to James Shapiro for
fruitful discussions. This work was supported by the Spanish
Centro de Astrobiologı́a (CSIC/INTA), associated with the
NASA Astrobiology Institute.
References
Aguilar C, Vlamakis H, Losick R & Kolter R (2007) Thinking
about B. subtilis as a multicellular organism. Curr Opin
Microbiol 10: 638–643.
Bai U, Mandic-Mulec I & Smith I (1993) SinI modulates the
activity of SinR, a developmental switch protein of Bacillus
subtilis, by protein–protein interaction. Genes Dev 7: 139–148.
Bobay BG, Mueller GA, Thompson RJ, Murzin AG, Venters RA,
Strauch MA & Cavanagh J (2006) NMR structure of AbhN and
comparison with AbrBN: FIRST insights into the DNA
binding promiscuity and specificity of AbrB-like transition
state regulator proteins. J Biol Chem 281: 21399–21409.
Branda SS, González-Pastor JE, Ben-Yehuda S, Losick R & Kolter
R (2001) Fruiting body formation by Bacillus subtilis. P Natl
Acad Sci USA 98: 11621–11626.
Branda SS, Chu F, Kearns DB, Losick R & Kolter R (2006) A
major protein component of the Bacillus subtilis biofilm
matrix. Mol Microbiol 59: 1229–1238.
Cao M, Wang T, Ye R & Helmann JD (2002) Antibiotics that
inhibit cell wall biosynthesis induce expression of the Bacillus
subtlis sW and sM regulons. Mol Microbiol 45: 1267–1276.
Chu F, Kearns DB, Branda SS, Kolter R & Losick R (2006) Targets
of the master regulator of biofilm formation in Bacillus subtilis.
Mol Microbiol 59: 1216–1228.
Chung JD, Stephanopoulos G, Ireton K & Grossman AD (1994)
Gene expression in single cells of Bacillus subtilis: evidence that
a threshold mechanism controls the initiation of sporulation.
J Bacteriol 176: 1977–1984.
Claverys JP & Håvarstein LS (2002) Extra-cellular peptide control
of competence for genetic transformation in Streptococcus
pneumoniae. Front Biosci 7: 1798–1814.
Claverys JP & Håvarstein LS (2007) Cannibalism and fratricide:
mechanisms and raisons d’être. Nat Rev Microbiol 5: 219–229.
Claverys JP, Prudhomme M & Martin B (2006) Induction of
competence regulons as general stress responses in Grampositive bacteria. Annu Rev Microbiol 60: 451–475.
Dagkessamanskaia A, Moscoso M, Hénard V, Guiral S, Overweg
K, Reuter M, Martin B, Wells J & Claverys JP (2004)
Interconnection of competence, stress and CiaR regulons in
Streptococcus pneumoniae: competence triggers stationary
FEMS Microbiol Rev 35 (2011) 415–424
phase autolysis of ciaR mutant cells. Mol Microbiol 51:
1071–1086.
Dubnau D & Losick R (2006) Bistability in bacteria. Mol
Microbiol 61: 564–572.
Eldholm V, Johnsborg O, Straume D, Ohnstad HS, Berg KH,
Hermoso JA & Håvarstein LS (2010) Pneumococcal CbpD is a
murein hydrolase that requires a dual cell envelope binding
specificity to kill target cells during fratricide. Mol Microbiol
76: 905–917.
Ellermeier CD, Hobbs EC, González-Pastor JE & Losick R (2006)
A three-protein signalling pathway governing immunity to a
bacterial cannibalism toxin. Cell 124: 549–559.
Errington J (2003) Regulation of endospore formation in B.
subtilis. Nat Rev Microbiol 1: 117–126.
Fawcett P, Eichenberger P, Losick R & Youngman P (2000) The
transcriptional profile of early to middle sporulation in
Bacillus subtilis. P Natl Acad Sci USA 97: 8063–8068.
Fujita M & Losick R (2003) The master regulator for entry into
sporulation in Bacillus subtilis becomes a cell-specific
transcription factor after asymmetric division. Genes Dev 17:
1166–1174.
Fujita M, González-Pastor JE & Losick R (2005) High- and lowthreshold genes in the Spo0A regulon of Bacillus subtilis.
J Bacteriol 187: 1357–1368.
Garcı́a P, González MP, Garcı́a E, Garcı́a JL & López R (1999) The
molecular characterization of the first autolytic lysozyme of
Streptococcus pneumoniae reveals evolutionary mobile
domains. Mol Microbiol 33: 128–138.
Garrido MC, Herrero M, Kolter R & Moreno F (1988) The export
of the DNA replication inhibitor Microcin B17 provides
immunity for the host cell. EMBO J 7: 1853–1862.
Gaur NK, Oppenheim J & Smith I (1991) The Bacillus subtilis sin
gene, a regulator of alternate developmental processes for a
DNA-binding protein. J Bacteriol 173: 678–686.
González-Pastor JE (2006) Multicellular and social behavior in
Bacillus subtilis. Bacillus: Cellular and Molecular Biology
(Graumann P, ed), pp. 423–453. Caister Academic Press,
Wymondham, UK.
González-Pastor JE, Hobbs EC & Losick R (2003) Cannibalism by
sporulating bacteria. Science 301: 510–513.
Guiral S, Mitchell TJ, Martin B & Claverys JP (2005)
Competence-programmed predation of noncompetent cell in
the human pathogen Streptococcus pneumoniae: genetic
requirements. P Natl Acad Sci USA 102: 8710–8715.
Hamon MA & Lazazzera BA (2001) The sporulation
transcription factor Spo0A is required for biofilm
development in Bacillus subtilis. Mol Microbiol 42: 1199–1209.
Håvarstein LS, Martin B, Johnsborg O, Granadel C & Claverys JP
(2006) New insights into the pneumococcal fraticide:
relationship to clumping and identification of a novel
immunity factor. Mol Microbiol 59: 1297–1307.
Johnsborg O, Eldholm V, Bjørnstad ML & Håvarstein LS (2008) A
predatory mechanism dramatically increases the efficiency of
lateral gene transfer in Streptococcus pneumoniae and related
commensal species. Mol Microbiol 69: 245–253.
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
424
Kearns DB, Chu F, Branda SS, Kolter R & Losick R (2005) A
master regulator for biofilm formation by Bacillus subtilis. Mol
Microbiol 55: 739–749.
Klein C & Entian KD (1994) Genes involved in self-protection
against the lantibiotic subtilin produced by Bacillus subtilis
ATCC 6633. Appl Environ Microb 60: 2793–2801.
Lewis RJ, Branningan JA, Offen WA, Smith I & Wilkinson AJ
(1998) An evolutionary links between sporulation and
prophage induction in the structure of a repressor: antirepressor complex. J Mol Biol 283: 907–912.
Lin D, Qu LJ, Gu H & Chen Z (2001) A 3.1-kb genomic fragment
of Bacillus subtilis encodes the protein inhibiting growth of
Xanthomonas oryzae pv. oryzae. J Appl Microbiol 91:
1044–1050.
López D, Fischbach MA, Chu F, Losick R & Kolter R (2009a)
Structurally diverse natural products that cause potassium
leakage trigger multicellularity in Bacillus subtilis. P Natl Acad
Sci USA 106: 280–285.
López D, Vlamakis H, Losick R & Kolter R (2009b) Cannibalism
enhances biofilm development in Bacillus subtilis. Mol
Microbiol 74: 609–618.
Luo P & Morrison DA (2003) Transient association of an
alternative sigma factor, ComX, with RNA polymerase during
the period of competence for genetic transformation in
Streptococcus pneumoniae. J Bacteriol 185: 349–358.
Michiels J, Dirix G, Vanderleyden J & Xi C (2001) Processing and
export of peptide pheromones and bacteriocins in Gramnegative bacteria. Trends Microbiol 9: 164–168.
Molle V, Fujita M, Jensen ST, Eichenberger P, González-Pastor JE,
Liu JS & Losick R (2003) The Spo0A regulon of Bacillus
subtilis. Mol Microbiol 50: 1683–1701.
Nakano MM, Magnuson R, Myers A, Curry J, Grossman AD &
Zuber P (1991a) srfA is an operon required for surfactin
production, competence development, and efficient
sporulation in Bacillus subtilis. J Bacteriol 173: 1770–1778.
Nakano MM, Xia LA & Zuber P (1991b) Transcription initiation
region of the srfA operon, which is controlled by the comPcomA signal transduction system in Bacillus subtilis. J Bacteriol
173: 5487–5493.
Nandy SK, Bapat PM & Venkatesh KV (2007) Sporulating
bacteria prefer predation to cannibalism in mixed cultures.
FEBS Lett 581: 151–156.
Nariya H & Inouye M (2008) MazF, an mRNA interferase,
mediates programmed cell death during multicellular
Myxococcus development. Cell 132: 55–66.
Parker GF, Daniel RA & Errington J (1996) Timing and genetic
regulation of commitment to sporulation in Bacillus subtilis.
Microbiology 142: 3445–3452.
Pei J & Grishin NV (2001) Type II CAAX prenyl endopeptidases
belong to a novel superfamily of putative membrane-bound
metalloproteases. Trends Biochem Sci 26: 275–277.
Perego M, Glaser P, Minutello A, Strauch MA, Leopold K &
Fischer W (1995) Incorporation of D-alanine into lipoteichoic
2010 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
J.E. González-Pastor
acid and wall teichoic acid in Bacillus subtilis. Identification of
genes and regulation. J Biol Chem 270: 15598–15606.
Peterson SN, Sung CK, Cline R et al. (2004) Identification of
competence pheromone responsive genes in Streptococcus
pneumoniae by use of DNA microarrays. Mol Microbiol 51:
1051–1070.
Piggot P & Losick R (2002) Sporulation genes and
intercompartmental regulation. Bacillus subtilis and Its Closest
Relatives: From Genes to Cells (Sonenshein AL, Hoch JA &
Losick R, eds), pp. 483–517. ASM Press, Washington, DC.
Piggot PJ & Coote JG (1976) Genetics aspects of bacterial
endospore formation. Bacteriol Rev 40: 908–962.
Prudhomme M, Attaiech L, Sanchez G, Martin B & Claverys JP
(2006) Antibiotic stress induces genetic transformability in the
human pathogen Streptococcus pneumoniae. Science 313:
89–92.
Shapiro JA (1998) Thinking about bacterial populations as
multicellular organisms. Annu Rev Microbiol 52: 81–104.
Sonenshein AL (2000) Endospore-forming bacteria: an overview.
Prokaryotic Development (Brun YV & Shimkets J, eds) pp.
133–150. ASM Press, Washington, DC.
Steinmoen H, Kenutsen E & Håvarstein LS (2002) Induction of
natural competence in Streptococcus pneumoniae triggers lysis
and DNA release from a subfraction of the cell population. P
Natl Acad Sci USA 99: 7681–7686.
Steinmoen H, Teigen A & Håvarstein LS (2003) Competenceinduced cells of Streptococcus pneumoniae lyse competencedeficient cells of the same strain during cocultivation. J
Bacteriol 185: 7176–7183.
Turner MS & Helmann JD (2000) Mutations in multidrug efflux
homologs, sugar isomerases, and antimicrobial biosynthesis
genes, differentially elevate activity of the sX and sW factors in
Bacillus subtilis. J Bacteriol 182: 5202–5210.
Varon M, Cohen S & Rosenberg E (1984) Autocides produced by
Myxococcus xanthus. J Bacteriol 160: 1146–1150.
Veening JW, Hamoen LW & Kuipers OP (2005) Phosphatases
modulate the bistable sporulation gene expression pattern in
Bacillus subtilis. Mol Microbiol 56: 1481–1494.
Vlamakis H, Aguilar C, Losick R & Kolter R (2008) Control of cell
fate by the formation of an arquitecturally complex bacterial
community. Genes Dev 22: 945–953.
Wireman JW & Dworkin M (1977) Developmentally induced
autolysis during fruiting body formation by Myxococcus
xhantus. J Bacteriol 129: 796–802.
Yao F & Strauch MA (2005) Independent and interchangeable
multimerization domains of the AbrB, Abh, and SpoVT global
regulatory proteins. J Bacteriol 187: 6354–6362.
Zheng G, Yan LZ, Vederas JC & Zuber P (1999) Genes of the sboalb locus of Bacillus subtilis are required for production of the
antilisterial bacteriocin subtilosin. J Bacteriol 181: 7346–7355.
Zheng G, Hehn R & Zuber P (2000) Mutational analysis of the
sbo-alb locus of Bacillus subtilis: identification of genes
required for subtilosin production and immunity. J Bacteriol
182: 3266–3273.
FEMS Microbiol Rev 35 (2011) 415–424