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Cannibalism by Sporulating Bacteria

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Science  25 Jul 2003:
Vol. 301, Issue 5632, pp. 510-513
DOI: 10.1126/science.1086462

Abstract

Spore formation by the bacterium Bacillus subtilis is an elaborate developmental process that is triggered by nutrient limitation. Here we report that cells that have entered the pathway to sporulate produce and export a killing factor and a signaling protein that act cooperatively to block sister cells from sporulating and to cause them to lyse. The sporulating cells feed on the nutrients thereby released, which allows them to keep growing rather than to complete morphogenesis. We propose that sporulation is a stress-response pathway of last resort and that B. subtilis delays a commitment to spore formation by cannibalizing its siblings.

Some microorganisms respond to nutritional limitation by entering a resting state in which they remain inactive for an extended time. Bacillus subtilis produces a robust resting cell, the endospore, that can remain dormant for many years. Endospore formation is an elaborate and energy intensive process that requires several hours to complete (14). If during this period nutrients were once again to become plentiful, the sporulating cells would be at a disadvantage relative to cells able to resume growth rapidly. Thus, bacteria could be expected to delay spore formation until forced to do so by prolonged depletion of nutrients. Here we present evidence that cells that have entered the pathway to sporulate delay development by killing their siblings and feeding on the nutrients thereby released. Cannibalism is mediated by an extracellular killing factor and a novel intercellular signaling protein that act cooperatively to cause cell death and impede sporulation.

Entry into sporulation is governed by the regulatory protein Spo0A (5). While building mutants of genes under the control of Spo0A (6), we discovered two operons (Fig. 1A) that are strongly induced at the start of sporulation (fig. S1) and in which mutations accelerated spore formation (Fig. 1, B and C, and fig. S2). We refer to these operons as skf for sporulation killing factor and sdp for sporulation delaying protein.

Fig. 1.

Mutants of the skf and sdp operons sporulate rapidly. (A) Gene organization of the skfAB CDEFGH and the sdpABC operons [previously annotated as ybcOPST ybdABDE and as yvaWXY, respectively (23)]. The hairpin symbols represent transcriptional terminators. (B) Colonies of skf [Δ(skfABCDEF)::tet; strain EG168] and sdp [Δ(sdpABC)::spc; strain EG407] mutants, as well as the double skf sdf mutant (EG523), were brighter (an indication of spore formation, see fig. S2) than those formed by the wild-type strain (PY79) after 14 hours of incubation on solid sporulation medium. Strains and plasmids used in our experiments are listed in table S2. (C) Time course of spore formation in solid medium by the wild-type (⚫), and the skf (▲), sdp (◼) and skf sdp (♢) mutants. The percentage of heat-resistant, colony-forming units (spores) versus total viable cells was monitored at the indicated times after inoculation in solid sporulation medium.

Clues that the eight-gene skf operon directs the production of an exported killing factor came from the similarity of its gene products to proteins involved in the production of peptide antibiotics (79). The first gene, skfA, encodes a small peptide, a characteristic of operons involved in the production of peptide antibiotics (7). The product of the second gene, skfB, is similar to a B. subtilis protein involved in the production of an antilisterial peptide, subtilosin (10). Finally, the product of skfD contains a domain characteristic of the CAAX family of amino terminal proteases (11, 12). The operon also contains two genes, skfE and skfF, whose products resemble an ATP–binding cassette transport complex (ABC transporter) and could be responsible for exporting the peptide antibiotic and conferring resistance to it.

To investigate these possibilities, we asked whether wild-type cells would kill cells of a mutant of the skf operon that had been marked with a lacZ fusion. Mutant and wild-type cells were mixed in equal proportions and grown in liquid sporulation medium. The ratio of mutant to wild-type cells remained approximately constant during growth but dropped dramatically after the onset of sporulation (Fig. 2A). These results indicate that the skf operon is involved in the production of an extracellular killing factor during sporulation. The operon must also confer resistance to the factor, because the mutation rendered cells sensitive to it. In keeping with these ideas, cells engineered to express the skf operon during growth in response to IPTG (isopropyl β-D-1-thiogalactopyranoside) caused killing when spotted on a lawn of wild-type or skf mutant cells and did so in a manner that was dependent on the presence of the IPTG inducer (Fig. 2B). Evidence that skfE and skfF encode an export pump for the killing factor came from placing the genes under the control of an IPTG-inducible promoter and introducing the construct into a strain that lacked the skf operon. The mutant cells were mixed with wild-type cells (tagged with lacZ) and grown in liquid sporulation medium. The number of mutant cells dropped sharply upon entry into sporulation when grown in the absence of IPTG but not when grown in its presence (Fig. 2C).

Fig. 2.

The skf operon produces a sporulation killing factor. (A) skf mutant cells harboring a lacZ fusion [Δ(skfABCDEF)::tet amyE::cotD-lacZ; EG169] and wild-type cells (PY79) were mixed in equal proportions and grown in liquid sporulation medium. The ratio of mutant to wild-type cells was determined at the indicated times before and after the start of sporulation (hour 0, ◼). As a control, ratios were determined for wild-type cells that had been mixed with wild-type cells that carried a lacZ fusion (strain PE29, ⚫) and for skf mutant cells (EG168) that had been mixed with skf mutant cells that carried a lacZ fusion (EG169, ▲). In these mixed cultures, the total number of viable cells during the time course was similar to that for individual cultures of the wild type or the skf mutant shown in (D). (B) Cells harboring the skf operon under the control of an IPTG-inducible promoter (strain EG208) were spotted on a lawn of wild-type or skf mutant cells growing on a rich (nonsporulation, Luria broth) medium. The engineered cells produced a halo of growth inhibition (arrow) in the presence (+) but not in the absence (–) of the inducer (1 mM IPTG). (C) Cells lacking the skf operon but containing a copy of skfE and skfF under the control of an IPTG-inducible promoter (strain EG219) were mixed in equal proportion with wild-type cells that carried a lacZ fusion (PE29), and the cell mixture was grown in liquid sporulation medium in the absence (◼) or in the presence (⚫) of the inducer (IPTG). The ratio of cells of strain EG219 to the cells of strain PE29 was determined at the indicated times after the start of sporulation. (D) Number of viable cells was measured in cultures of wild-type cells (⚫), and cells of skfA (EG165) (◼) and skfABCDEF (EG168) (▲) mutants in liquid sporulation medium at the indicated times after the start of sporulation.

We next asked whether the operon causes death in a homogenous population of wild-type cells. Cultures of cells sporulating in liquid medium show a characteristic drop in optical density shortly after the start of spore formation. This drop was associated with a dramatic (∼70%) decrease in the number of viable cells, and, of note, in a manner that was dependent on skf (Fig. 2D and fig. S3). The simplest interpretation of these results is that the wild type produces a mixed population in which Spo0A is active (and directing transcription of skf) in some cells and not in others [fig. S4 and (13, 14)]. Cells with active Spo0A would produce the killing factor and the pump that exports it. Cells with inactive Spo0A would produce neither the factor nor the pump, and they would be killed. Thus, the killing factor is responsible for killing genetically identical cells (siblings) in the population. This is contrary to the traditional paradigm of chemical warfare among microorganisms in which antibiotics are used to kill other, competing species.

Why do colonies of skf mutant cells exhibit accelerated sporulation (15)? We suggest that the killing factor causes cells in which Spo0A is inactive to lyse and release nutrients, which allow cells in which Spo0A is active but which have not yet committed to morphogenesis to keep growing. An skf mutant, in contrast, does not cause killing and hence sporulation takes place without delay.

Mutations in a second operon, sdp (Fig. 1A), also caused an accelerated sporulation phenotype and did so more rapidly than mutations in skf (Fig. 1C and fig. S2). What is the mechanism by which this three-gene operon delays spore formation? To answer this question, we carried out microarray analysis to identify genes whose transcription was dependent on the operon (fig. S5 and table S1). Two genes whose transcription was strongly dependent on sdp were yvbA (whose inferred product is similar to the ArsR family of transcriptional regulators) and yvaZ (whose product is of unknown function but is inferred to contain multiple transmembrane segments). The yvbA and yvaZ genes constitute an apparent operon that is located immediately downstream of, and in convergent orientation to, the sdp operon itself (Fig. 1A). The use of lacZ fused to the promoter for yvbA and yvaZ (PyvbA yvaZ-lacZ) confirmed that transcription of the operon was almost completely dependent on sdp (Fig. 3A).

Fig. 3.

The sdp operon encodes an extracellular signaling protein. (A) Wild-type cells and sdp mutant cells containing a PyvbA-yvaZ-lacZ fusion (EG381 and EG524, respectively) were grown on solid sporulation medium containing X-gal (5-bromo-4-chloro-3-indolyl B-d-galactopyranoside). (B) Cells of the sdp mutant harboring PyvbA-yvaZ-lacZ (EG524, white arrows) were streaked on solid sporulation medium in the vicinity of a streak of wild-type (strain PY79, left) or sdp mutant cells (EG407, right). (C) Eluates from reversed-phase chromatography of supernatant fluids from cultures of wild-type and sdp mutant (EG407) cells (at hour 1.5 of sporulation in liquid medium) were subjected to SDS–polyacrylamide electrophoresis in a 4 to 20% gradient gel. (D) Supernatant fluids were collected from cells of a strain (EG351) in which the sdp operon was under the control of an IPTG-inducible promoter and grown in the absence (⚫) or in the presence (▲) of the inducer (IPTG). Eluates from reversed-phase chromatography of the supernatant fluids from the cultures were added to cells of an sdp mutant harboring PyvbA-yvaZ-lacZ (EG524) growing in minimal medium. Culture samples were collected at the indicated times and assayed for β-galactosidase activity.

Remarkably, this dependence was mediated by intercellular signaling. Expression of PyvbA yvaZ-lacZ in cells mutant for sdp was restored when the mutant cells were grown in close proximity to wild-type cells on solid medium (Fig. 3B). No restoration of lacZ expression was observed when the mutant was grown close to cells mutant for sdp. Evidently, sdp is responsible for the production of an extracellular factor that is capable of inducing the transcription of yvbA and yvaZ in recipient cells. We purified from conditioned medium from a culture of wild-type cells a fraction containing a ∼5-kD protein that stimulated β-galactosidase synthesis when added to cells of an sdp mutant that harbored PyvbA yvaZ-lacZ [Fig. 3C and (16)]. Neither the stimulatory activity nor the protein was present in conditioned medium from sdp mutant cells. A protein of similar size was obtained with cells engineered to express the sdp operon during growth in response to IPTG. Again, the fraction containing this protein stimulated β-galactosidase production (Fig.3D). Finally, sequential Edman degradation (-GLYAV-VAAGYLYVVGVNAALQTAAAV) (12) revealed that the ∼5-kD protein originated from the product of the sdpC gene of the operon, its N-terminal residue corresponding to residue 141 of the 203residue-long protein.

Next, we asked whether induction of the yvbA yvaZ operon, and yvbA in particular, was responsible for the delay in sporulation caused by the signaling protein by engineering cells to express yvbA or yvaZ or both in response to IPTG. The results show that artificial induction of yvbA and yvaZ or of yvbA alone (but not yvaZ alone) was sufficient to delay sporulation (Fig. 4A). Transcriptional profiling with cells mutant for the sdp operon revealed candidates for genes that could be under the control of the YvbA transcription factor (above; fig. S5 and table S1). Among these were the ATP synthase operon (atpIBEFHAGDC), which is responsible for ATP production, and the yusLKJ operon, whose inferred products are similar to lipid catabolism enzymes (fig. S5 and table S1). Use of lacZ fused to yusLKJ confirmed that high-level expression of the operon was dependent on the signaling protein and on YvbA (Fig. 4B). Also, artificial induction of YvbA synthesis restored the expression of yusLKJ to cells doubly mutant for the sdp and yvbA yvaZ operons (Fig. 4B). We propose that the signaling protein turns on the synthesis of YvbA, which, in turn, causes an increase in lipid oxidation and ATP production. The proposed increase in energy production could be responsible for delaying sporulation, which is triggered by depletion of energy reserves.

Fig. 4.

The effect of the sdp-encoded signaling protein is mediated by the putative transcription factor YvbA. (A) Overexpression of yvbA yvaZ or yvbA delays sporulation in a strain lacking the sdp and the yvbA yvaZ operons. Constructs were created in which either yvbA and yvaZ or yvbA alone or yvaZ alone were under the control of an IPTG-inducible promoter (Pspac-hy) and introduced into a strain, EG494, that was mutant for sdpABC and yvbA yvaZ. EG494 and its derivatives were grown on solid sporulation medium in the absence and in the presence of IPTG: 1, EG494; 2, a derivative of EG494 harboring Pspac-hy-yvbA yvaZ (EG525); 3, a derivative of EG494 harboring Pspac-hy-yvbA (EG526); and 4, a derivative of EG494 harboring Pspac-hy-yvaZ (EG527). The wild type was strain PY79. (B) Time course of accumulation of β-galactosidase from PyusLKJ-lacZ in a wild-type strain (EG447) (⚫), and in a strain (EG484) mutant for sdp and yvbA yvaZ and harboring Pspac-hy-yvbA. The cells were grown in the absence (▲) or presence of 1 mM IPTG (◼). Culture samples were collected at the indicated times before and after the start of sporulation (hour 0). (C) Time course of the number of viable cells during sporulation of a strain (EG526) mutant for sdp and yvbA yvaZ and harboring Pspac-hy-yvbA (⚫◯) and of a derivative of EG526 that was additionally mutant for sfk (EG528) (◼▢) grown in the absence (open symbols) and in the presence of 1 mM IPTG (filled symbols).

Finally, and coming full circle, we found that artificial induction of YvbA synthesis caused a marked drop in cell viability in a manner that was dependent on the skf operon (Fig. 4C). Evidently, synthesis of the YvbA transcription factor causes enhanced sensitivity to the sporulation killing factor. It could do so by stimulating the expression of genes involved in energy production, as metabolically active cells are more sensitive to antibiotics than are quiescent cells (17, 18). Also, yvbA was previously identified in a screen for genes that inhibit the expression of the gene for σW, a regulatory protein that turns on genes involved in detoxification and resistance to antibiotics (16, 19, 20). Thus, YvbA-mediated repression of the gene for σW could heighten sensitivity to the killing factor by suppressing the antibiosis stress response.

We conclude that sporulating cells of B. subtilis are cannibalistic, feeding on their siblings in order to delay committing to spore formation. Because sporulation becomes irreversible after its earliest stage, delaying spore formation as long as possible might be beneficial, as a cell that is committed to spore formation could be at a disadvantage relative to other cells should nutrient deprivation prove to be fleeting. Wild (but not laboratory) strains have been found to assemble into multicellular structures in which spore formation preferentially takes place at the apical tips (21). Perhaps the killing factor and signaling protein influence the timing and localization of spore formation in these fruiting-body-like structures. Fruiting body formation by the unrelated spore-forming bacterium Myxococcus xanthus is reported to involve lysis of nonsporulating cells (22). Conceivably, this killing is mediated by cells in the developing fruiting body that have entered the pathway to sporulate. It will be interesting to see whether the killing of genetically identical siblings is a widespread feature of the dynamics of bacterial populations.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1086462/DC1

Material and Methods

SOM Text

Figs. S1 to S5

Tables S1 and S2

References

References and Notes

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