() have demonstrated that sporulating bacteria prefer predation of other microorganisms to cannibalism in mixed cultures. Cells from stationary cultures of . Bacteria, thus, delay spore formation, since if nutrients were to be once again available, the sporulating cells would be at a disadvantage. kills and consumes its siblings to survive starvation and delay sporulation.
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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: Sporulating cells produce caninbalism 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 bacteriw 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.
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. Unexpectedly, it has been reported that some members of a genetically identical population can kill their siblings using antimicrobials.
In the case of Bacillus subtiliscells 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 Cannnibalism. In Streptococcus pneumoniaa fratricide behavior was described in which bcateria that are competent for natural genetic transformation express proteinaceous toxins that will lyse noncompetent siblings Steinmoen et al.
A common feature of this phenomenon in these microorganisms is sporulatinh it is based on a differentiation process: Bacillus subtilis cells enter into sporulation under nutrient limitation conditions.
Spo0A, the master sporulatinng of sporulation, becomes active only in a part of the population. The Spo0A-active cells produce and are immune to the Skf and Sporhlating toxins. The Spo0A-inactive cells are sensitive to the toxins because 1 the ABC transporter and other putative immunity genes involved in Skf cannibxlism are not transcribed and 2 AbrB is expressed in Spo0A-inactive cells abrB gene is repressed by Spo0Aand 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. The key stages of the cycle are shown. 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.
The mother cell engulfs the forespore, and contributes to the transformation of the forespore to spore, but ultimately the mother cell undergoes lysis Fig.
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. 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. 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.
The killing of nonsporulating cells is controlled by two independent gene clusters Fig. Both clusters were previously identified as positively regulated by Spo0A, the master regulator of sporulation Fawcett et al.
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 hairpin symbols represent transcriptional terminators.
The skf operon contains eight genes skfA-HFig. Finally, the products encoded by skfE and skfF resemble the two components of an ATP-binding cassette transport complex ABC transporterthe 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. 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.
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. 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. 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: The sdpABC operon is responsible for the production and secretion of a amino-acid protein that is derived from the C-terminal portion of the product of sdpC.
Recently, SdpC has been shown to be a toxin, and the products of sdpRI confer immunity to the toxin Ellermeier et al.
Cannibalism by Sporulating Bacteria
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. Competition experiments with mixtures of wild-type and sdp mutant cells support the role of SdpC as a toxin. SdpR directly regulates the sdpRI operon zporulating 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 sdpRwhich 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.
Furthermore, sdpI mutants blocked in an activated state for sdpRI transcription in the absence of the SdpC signal and sdpI mutants unable to induce a transcriptional sporulatinb in the presence of SdpC were obtained, and both types of mutants retain the capacity to confer immunity.
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. Model of how SdpC induces expression of the sdpRI immunity operon. 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. The cannibalism in sporulating cultures is a consequence of the population heterogeneity regarding Spo0A activation.
Bacteira has been reported that isogenic bacteria grown under identical conditions do not display the same pattern of gene expression, which is termed bistability. In a culture of sporulating B. The auto-stimulatory loops controlling Spo0A activity of spo0A are involved in the generation of a bistable response Veening et al. Therefore, the killing factors and the resistance to it cannibaism be produced by Spo0A-active cells, but not by Spo0A-inactive cells, which will be killed.
Why are the sporulating cells immune to the toxins that they produce? Immunity vy 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. 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 skfDwhose product contains a domain that shows homology to the type II CAAX prenyl endopeptidases. The sporulating cells are also protected from SdpC, because the immunity protein SdpI is induced in the presence of SdpC.
Cannobalism, 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. Sporulatting 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. 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 sporylating cellsin order to relieve repression through AbrB. The accelerated sporulation phenotype of the skf and sdp mutants reveals that the sporulation process in B.
The killing factors produced by these operons at the first signals of starvation lyse nonsporulating cells, which are sensitive to killing. Then, bactdria are released into the medium, and those cells in which Spo0A is active, but which have not yet committed to sporulation, can 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. Activated Spo0A directly controls genes organized in 30 single genes and 24 operons or clusters Fawcett et al. 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.
More recently, transcriptional-profiling experiments using DNA microarrays were applied to identify the Spo0A-dependent genes distributed in each category Fujita et al. It was observed that genes requiring a high dose of Spo0A to be activated have a low binding affinity for sporulatlng regulator.
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.
Cannibalism: the bacterial way
The operons involved in cannibalism, skf and sdpABCfall 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.
Moreover, AbrB directly represses transcription of the skf operon; thus, skf is activated by two routes: 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.
The building of these multicellular structures can be seen as a prelude to spore formation. Then, if 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. Different levels of active Spo0A Spo0A phosphorylated, Spo0A-P control cannibalism, formation of multicellular structures and sporulation.