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The phosphorelay integrates both positive and negative signals, ensuring that sporulation, a time- and energy-consuming process that may bring an ecological cost, is only triggered should other adaptations fail. Here we report that a gastrointestinal isolate of B. Sporulation during growth occurs because Spo0A is more active per cell and in a higher fraction of the population than in a laboratory strain.
This in turn, is primarily caused by the absence from the gut strain of the genes rapE and rapK , coding for two aspartyl phosphatases that negatively modulate the flow of phosphoryl groups to Spo0A. We show, in line with recent results, that activation of Spo0A through the phosphorelay is the limiting step for sporulation initiation in the gut strain. Our results further suggest that the phosphorelay is tuned to favor sporulation during growth in gastrointestinal B.
Bacillus subtilis is a naturally competent, spore-forming Gram-positive bacterium that can be isolated from many aquatic and terrestrial environments. The capacity to develop highly resistant endospores in response to nutrient scarcity, allied with their dispersal, explains in part the isolation of this organism from a wide range of environmental samples 1 , — 3. However, evidence also suggests that B. In particular, its recurrent isolation from the gut of several invertebrates and vertebrates, including humans 9 , suggests that B.
Spores appear to be important for the ability of B. Spores resist digestion by protozoa and passage through the GI tract of the mouse 10 , Moreover, spores are also able to germinate and undergo new cycles of growth and sporulation in the GI tract of mice The ubiquitous nature of B.
As more strains of this species are sequenced and studied, it is expected that niche-specific genes will be identified 3 , In the laboratory, spore formation by B. Sporulation is triggered after cells enter the stationary phase in a medium that supports sporulation, and it takes about 8 to 10 h to be completed 13 , Central to the decision to enter sporulation is the activation, by phosphorylation, of the response regulator Spo0A.
Transfer of phosphoryl groups to Spo0A occurs via a series of components called the phosphorelay, served by five sensor kinases KinA to -E that autophosphorylate at a histidine residue 15 , — 19 Fig. Phosphate is then transferred to an aspartate residue of the Spo0F response regulator and finally to an aspartate of the response regulator domain of Spo0A, via a histidine residue in the Spo0B phosphotransferase 13 , 15 , 17 , 19 Fig.
Thus, cells will attempt several responses to starvation, prior to, as a last resource, and upon embarking into the developmental pathway of sporulation 25 , Activation of Spo0A and the initiation of sporulation. A Simplified representation of the circuits involved in the initiation of sporulation in B. The figure also illustrates the actions of the RapA phosphatase and the PhrA peptide.
Panel B represents a simplified version of the noise generator model that accounts for the generation of heterogeneity in the levels of phosphorylated Spo0A during entry into the stationary phase. During the transition from exponential growth to stationary phase, starvation-sensing pathways 35 , 37 , 38 converge with quorum-sensing pathways via the Rap-Phr family of phosphatases to regulate the phosphorylation of Spo0A Noise generation by the phosphorelay results from fluctuations in the levels of expression of the genes for the phosphorelay components 40 , 41 and also from the action of the Rap phosphatases 42 , — 44 Fig.
Deletion of the genes for the Rap phosphatases reduces noise and allows for increased, more synchronized sporulation 42 , Since spore differentiation in B. In this investigation, we characterized the sporulation of an undomesticated isolate of B. We show that BSP1 initiates sporulation during growth and hence bypasses the cell density, nutritional, and other signals that normally restrain the flow of phosphoryl groups to Spo0A and delay sporulation. We show that sporulation is initiated during growth because of increased activation of Spo0A and that this primarily results from the absence of the genes for two Rap phosphatases.
Our results also suggest that the increased activation of Spo0A is carefully balanced in BSP1, so that growth is not compromised. Because different combinations of Rap phosphatases appear to be absent from other gut strains of B. Luria-Bertani LB medium was used for the routine growth of both B. Sporulation of B. For B. Normalization was achieved by adjusting all ratios so that the median of all log 2 ratios for a given experiment was zero The B.
Plasmid construction is also described in the supplemental material and listed in Table S2 in the supplemental material. Oligonucleotide primers used for PCR, mutagenesis, or sequencing are listed in Table S3 in the supplemental material. Competent cells of B. For mixed experiments see Fig. Quantification of the signal from P spo0A - yfp Fig. Around cells were analyzed for each strain at each time point tested. Quantification of the green fluorescent protein GFP signal see Fig.
The cell mixture was then stained with the membrane dye FM and observed by phase-contrast and fluorescence microscopy. The first column shows phase-contrast images, the middle column shows images of the same cells stained with DAPI and FM, and the right column shows P spo0A - yfp expression.
Vegetative cells of BSP1 derivative cells labeled with arrowheads show stronger spo0A-yfp expression at all times compared with the same cells in the PY79 background cells labeled with stars. The quantification was based on images such as those in panel A; the cells scored in each class y axis did not show morphological signs of sporulation see Materials and Methods. The titer of heat-resistant spores was measured at the indicated times in hours before or after the onset of sporulation.
The results shown are the averages of results from three independent experiments with error bars representing the standard deviation. The various cultures were grown in DSM, and samples were collected at the onset of sporulation T 0 for fluorescence microscopy and quantitative analysis Materials and Methods. C Percentage of cells with no morphological signs of sporulation predivisional cells and with a visible asymmetric septum or other morphological features indicative of later stages of sporulation postdivisional cells for the indicated strains.
In all cases, samples of 10 ml were collected. When grown on sporulation plates DSM , strain BSP1 formed opaque colonies, a symptom of spore formation 22 , faster than a reference laboratory strain such as PY79 did not shown.
We used phase-contrast and fluorescence microscopy to examine progress through the morphological stages of sporulation for derivatives of both strains bearing a ywcE-gfp translational fusion. The ywcE gene codes for a holin-like protein that localizes to the cell and spore membranes, allowing their visualization throughout sporulation The results in Fig.
In contrast, such morphogenetic signs were only detected for the PY79 derivative 2 h after T 0. This suggested that BSP1 initiated sporulation during growth. To have a more quantitative description of the process of sporulation, we measured the titer of heat-resistant spore formation for the two strains when grown on liquid DSM. The data are consistent with the conclusion that morphogenesis proceeded at the same pace for the two strains see Fig.
S1A in the supplemental material. Also, in agreement with the inference that BSP1 initiated sporulation efficiently during growth, when late-log-phase BSP1 cultures were diluted, the spore titer reached the original, predilution number in about 2 h see Fig. In contrast, for PY79, the number of spores did not increase following dilution. This implies the presence in BSP1 cultures of a high proportion of spores close to the stage of becoming heat resistant see Fig.
We conclude that BSP1 initiates sporulation during growth, bypassing the nutritional and cell density signals that normally delay sporulation until entry into the stationary phase. Sporulation during growth of BSP1. The cells were observed by phase-contrast PC and fluorescence FL microscopy.
YwcE-GFP labels the cell and spore membranes 48 and was used to visualize the spore membranes following completion of engulfment. Signs of asymmetric division ad , engulfment initiation ei , engulfment completion ec , phase gray pg , phase bright pb , and free spores sp are highlighted with arrowheads. The top row shows phase-bright or phase-gray spores, the middle row engulfment completion, and the bottom row asymmetric septation.
The quantification is based on the analysis of images like those of panel A see Materials and Methods. T 0 indicates the end of the exponential growth phase. The spore titer was measured at the indicated times in hours before or after T 0. Since sporulation initiation depends on the activation, by phosphorylation, of Spo0A 13 , we reasoned that perhaps this protein was active in a larger fraction of the BSP1 population of growing cells compared to the laboratory strain PY While this is most likely due to the high proportion of spores found for BSP1 early in the stationary phase, and from which proteins are more difficult to extract, it became clear that immunoblot analysis could not provide an adequate comparison between the levels of Spo0A between the two strains.
Moreover, Spo0A-yellow fluorescent protein YFP accumulates to high levels in the mother cell, following asymmetric division, further complicating the comparison between cells entering sporulation in the two strains This allowed the identification of cells in which asymmetric division had not yet occurred and the quantification of the YFP signal in those cells under the same viewing conditions.
Since Spo0A is autoregulatory, the fusion reports both transcription of spo0A as well as the activity of Spo0A In contrast, At the onset of the stationary phase T 0 , the percentages of cells above the threshold increased to The distribution of the YFP signal among the population of vegetative cells did not reveal a bistable pattern but rather was heterogeneous, in line with the conclusion of recent studies 42 , Also importantly, no differences were seen in the coding sequence or the regulatory region of the spo0A gene between BSP1 and PY79 not shown.
Thus, Spo0A reaches levels sufficient to trigger sporulation in a larger fraction of the BSP1 population reaching the stationary phase. We then wanted to determine whether the increased activity of Spo0A in the BSP1 strain could be attributed to augmented transcription of spo0A , which relies in part on the activation of Spo0A, as Spo0A is autoregulatory 21 , or to increased activation of Spo0A through the phosphorelay. To eliminate the autoregulatory loop acting at the level of spo0A transcription, we replaced the wild-type copy of the gene with a xylose-inducible P xylA - spo0A allele in BSP1 and PY79 derivatives additionally carrying a P spoIIG - cfp fusion.
We then monitored the accumulation of Spo0A and the level of sporulation as a function of the xylose concentration. Control experiments, in which we have used a P xylA - gfp fusion, have shown that the responses of the P xylA promoter to xylose are very similar in both BSP1 and PY not only GFP showed a parallel accumulation for each concentration of xylose tested for the two strains albeit at slightly lower levels for BSP1 , but the distribution of the fluorescence signal across the population was also very similar see Fig.
S2 in the supplemental material. Therefore, it appears that the accumulation of Spo0A is somehow limited in the background of BSP1 see also below. Importantly, for PY79, 0. Clearly, for a xylose concentration of 0. Furthermore, and in line with the conclusion of recent studies 42 , 43 , our results suggest that the activation of Spo0A and not its enhanced production through the positive autoregulatory loop that acts at the level of spo0A transcription is the foremost factor for entry into sporulation.
Also in agreement with this conclusion, inactivation of the P s promoter of the spo0A gene caused a fold reduction in the final titer of spores measured 24 h after the onset of the stationary phase in DSM formed by PY79 but only a 5-fold reduction in strain BSP1 see Fig. S3 in the supplemental material. Spo0A is more active per BSP1 cell. The bottom part of the panel shows the titer of heat-resistant spores for each of the cultures analyzed in panel A, measured 24 h after addition of xylose.
The red bars indicate the results obtained with 0. Samples were examined by phase-contrast PC and fluorescence microscopy 5 h after addition of xylose. Samples were collected and analyzed at the indicated times in minutes. In the experiments described above, extracts were prepared for immunoblot analysis 2 h after induction with xylose. Therefore, it seemed possible that Spo0A initially accumulated to high levels in BSP1, triggering sporulation, its levels then rapidly falling.
To test this possibility, we monitored the kinetics of accumulation of Spo0A after induction of cultures with 0. For PY79, the level of Spo0A increased with time until min after induction, reaching a plateau thereafter Fig. In BSP1, the level of Spo0A increased with time but always at lower levels than with PY79 , reaching a maximum min following induction, after which its levels receded Fig.
Because Spo0A is more active per BSP1 cell, the results suggest that a mechanism is in place to limit the accumulation of active Spo0A. As an independent test of this idea, we replaced the wild-type spo0A gene, in both PY79 and in BSP1, with a fusion of the P xylA promoter to the sad67 allele, which codes for a constitutively active form of Spo0A. The BSP1 and PY79 derivatives bearing the P xylA - sad67 construct additionally carried a P spoIIG - cfp fusion, so that transcription of an Spo0A-dependent sporulation gene could be monitored by microscopy in relation to spore morphogenesis.
For reference, the accumulation of Spo0A was inspected in cultures of BSP1 and PY79, to which xylose was added to the same concentration 0. Moreover, while in PY79 Spo0A accumulated to similar levels throughout the experiment, in BSP1 it reached a maximum 1 h after induction, and then its level decreased Fig. Consistent with the lack of expression of the high-threshold spoIIG operon, no asymmetric septa, the formation of which requires high levels of Spo0A, were seen in the sad67 derivative of BSP1 not shown.
Note that this strain, however, will reach a titer of spores identical to that of the PY79 derivative bearing the sad67 allele after 24 h of incubation. Together, these results suggest that in BSP1, increasing the activity of Spo0A above a certain threshold level triggers a pathway that leads to elimination of the transcription factor.
The unusual sporulation properties of strain BSP1—in particular, the observation that Spo0A reached sufficient levels of activity to promote sporulation during growth—prompted us to test whether genomic variation could be detected in the genes coding for the components of the phosphorelay. Using microarray-based comparative genomic hybridization M-CGH 12 , we found that spo0A , spo0B , and spo0F , as well as the genes kinA to - E coding for the five histidine kinases controlling the initiation of sporulation, were present and with no detectable variability in the genome of BSP1 compared to strain see Table S4 in the supplemental material.
Of note is the divergence found for the lrpA and lrpB genes, which function in a KinB stimulatory pathway We then tested whether activation of Spo0A still required the phosphorelay. KinA and KinB are the main kinases involved in the initiation of sporulation 42 , 50 , Therefore, while KinA retains its central role in the initiation of sporulation in BSP1, the results suggest that at least one other kinase is responsible for the residual sporulation of the kinA kinB double mutant.
This residual sporulation appears to be the responsibility of KinC, because inactivation of kinC in a kinA kinB double mutant but not the inactivation of kinD or kinE caused a fold reduction in the spore titer measured at h 24 Fig. This conclusion is in agreement with the results of a previous study that implicated KinC in a residual level of sporulation observed for laboratory strains during growth in minimal medium It is also in agreement with the finding that the overproduction of either KinA or KinC can markedly enhance the fraction of cells with polar septa, the formation of which requires the expression of high-threshold Spo0A-dependent genes Effect of the phosphorelay kinases on sporulation by BSP1.
The kinetics of heat-resistant spore formation was monitored for strains BSP1 black and PY79 white and derivatives of both strains bearing the indicated single or multiple deletions of the genes coding for the phosphorelay kinases kinA , kinB , kinC , kinD , and kinE.
The titer of heat-resistant spores was measured at the indicated times in hours after the onset of sporulation T 0 in DSM. The dashed lines indicate the levels of sporulation for the parental strains at T 0. These genes are located within or close to mobile genetic elements that may be absent from the genome of BSP1 see Table S4 and the supplemental material for a more detailed description of these results.
Also, PCR with primers designed on the basis of the genome sequence of strain see Fig. S4 in the supplemental material failed to amplify the rapE , rapI , and rapK genes. The rapI-phrI pair is also missing from the genome of the laboratory strain PY79 but present in other commonly used strains, such as MB24 data not shown. Because at least some of the five Rap phosphatases are known to drain phosphate from Spo0A during growth by dephosphorylating Spo0F and are antagonized by cognate inhibitory peptides in a cell density-dependent manner 28 , — 33 , we hypothesized that the absence of rapE , rapI and rapK could be related to the ability of BSP1 to initiate sporulation during growth.
We then examined the titer of heat-resistant spores in the various strains during entry into the stationary phase in a sporulation medium in comparison with BSP1. Cannibalism, which is mediated by the production of a killing factor coded for by the skf and sdp operons, is a well-studied mechanism for delaying sporulation 22 , Both the skf and sdp operons belong to the low-threshold class of Spo0A-dependent genes, and skf also codes for a transporter that confers immunity to the Skf producer cells.
Spo0A-inactive cells in which the skf operon is not transcribed are sensitive to the toxin and are killed, allowing the resistant cells to feed on the released nutrients and delay entry into sporulation 22 , 23 , S4 in the supplemental material confirmed the presence of the skf operon in BSP1. S5A in the supplemental material. However, and in agreement with a recent reannotation of the genome of strain 54 , we found that in both BSP1 and PY79, the skfC and skfD cistrons are fused into a single gene see Fig.
Neither of these manifestations was seen for BSP1, irrespective of whether the operon was deleted see Fig. S5C and D. S6A in the supplemental material. As previously reported 22 , cell lysis was strongly dependent on an intact skf locus for PY79 see Fig. For BSP1, however, lysis was independent of the presence of the skf operon see Fig. In any case, the results suggest that the skf -mediated delay of entry into sporulation previously documented for PY79 does not operate in BSP1.
Strain BSP1 shows robust induction of sporulation in part because it lacks the genes for three of the Rap phosphatases that are thought to help delay sporulation until the stationary phase of growth. Moreover, BSP1 is immune to the delay imposed upon entry into sporulation by expression of the skf operon. These observations motivated us to inspect other wild isolates of B. We used PCR, with primers designed based on the sequence of the strains, to test for the presence of the rap genes and of the skf operon in nine additional B.
In addition, six other B. In parallel, we examined a total of four laboratory strains, including PY While rapA was present in all strains tested, most of the strains lacked at least one rap gene. S4 in the supplemental material. S4 , our PCR assay generates false negatives. In contrast, poultry strain , which lacked only rapI , formed 4. The titer of spores measured at h 4 of the stationary phase in DSM was between 1.
The absence of rapE and phrE has been noticed in other isolates of B. The rapE-phrE operon is located within the skin element, which is also not strictly conserved in strains of B. Our analysis suggests a correlation between enhanced sporulation and the absence of certain Rap phosphatases. It also illustrates the plasticity of the B. In fact, this plasticity also extends to the skf operon, which seemed absent or was divergent from one of the strains of ovine or bovine origin, and from seven of the nine additionally tested strains derived from broiler chickens see Fig.
Here, we have analyzed entry into sporulation in a poultry GI tract isolate of B. In this strain, as also observed for B. Previous studies have indicated that RapE has an accessory role in sporulation However, the effect of deletion of rapE on the sporulation titer was assessed after overnight growth, when, even for BSP1, the differences compared to a laboratory strain are not as evident as during the earlier times of sporulation.
In contrast, the rapK-phrK operon, together with the rapC-phrC and rapF-phrF pairs, has been implicated in the expression of ComA-dependent genes 60 but not in sporulation. Likewise, the rapI-phrI system, which regulates mobilization of the integrative and conjugative element Bs1 , has not been connected to sporulation ICE Bs1 Presumably, the role of these operons in sporulation is restricted to a specific niche or genomic context and does not emerge under laboratory conditions for the commonly used strains.
In any event, because reintroduction of the rap operons in BSP1 does not reproduce exactly the kinetics of sporulation of the laboratory strain PY79, it is likely that other factors contribute to the initiation of sporulation during growth. Our results suggest that in BSP1, the phosphorelay is tuned to trigger sporulation during growth.
This contrasts with the behavior of laboratory strains, in which several adaptive responses are mounted during the transition to the stationary phase, and entry into sporulation is delayed. Cannibalism, for instance, is not manifested in strain BSP1. The expression of the operon gene skf , coding for the sporulation-killing factor, which mediates killing of Spo0A-inactive sister cells 22 , 23 , 53 , 62 , is detected in BSP1.
However, Skf-mediated lysis is not perceptible, presumably because of the large fraction of cells in which Spo0A accumulates and that are Skf resistant. Initiation of sporulation during growth may confer an adaptive advantage in the gut ecosystem. There are examples of spore-forming bacteria in which sporulation is central to their survival in the gut ecosystem. Metabacterium polyspora , for instance, is a strict anaerobe that lives in close association with its coprophagous host, the guinea pig 63 , In this organism, formation of multiple endospores, which will also protect the organism outside its host, is the primary form of propagation 63 , Only mature spores survive passage through the mouth and stomach of the guinea pig.
The spores will germinate in the intestine and will initiate the next round of sporulation, bypassing binary fission. Several species or morphotypes of Epulopiscium , an intestinal symbiont of the surgeonfish, are also known to use endospore formation as a form of survival and dispersion. The music is cautious, nervous, foreboding and fearful.
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