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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 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 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. 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 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 FEMS Microbiol Rev 35 (2011) 415–424 417 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 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 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. 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 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) 415–424 419 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 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 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 Published by Blackwell Publishing Ltd. All rights reserved c 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) 415–424 421 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 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 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 Published by Blackwell Publishing Ltd. All rights reserved 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. 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