Spore development and stress resistance in Bacillus subtilis are governed by the master transcription factors Spo0A and ^B, respectively . Here we show that the coding genes for both regulatory proteins are dramatically induced, during logarithmic growth, after a temperature downshift from 37 to 20°C . The lossof ^B reduces the stationary-phase viability of cold-adaptedcells 10- to 50-fold . Furthermore, we show that ^B activity isrequired at a late stage of development for efficient sporulationat a low temperature . On the other hand, Spo0A loss dramaticallyreduces the stationary-phase viability of cold-adapted cells10,000-fold . We show that the requirement of Spo0A for cellular survival during the cold is independent of the activity of the key transition state regulator AbrB and of the simple loss of sporulation ability . Furthermore, Spo0A, and not proficiencyin sporulation, is required for the development of completestress resistance of cold-adapted cells to heat shock [54°C,1 h], since a loss of Spo0A, but not a loss of the essentialsporulation transcription factor ^F, reduced the cellular survivalin response to heat by more than 1,000-fold . The overall resultsargue for new and important roles for Spo0A in the developmentof full stress resistance by nonsporulating cells and for ^B in sporulation proficiency at a low temperature.

 
The exposure of bacteria to diverse growth-limiting conditions induces the synthesis of a large set of proteins [called general stress proteins] that protect the cell against internal [metabolic] or external [environmental] stresses [22, 23, 29, 32, 33] . In the gram-positive, endospore-forming bacterium Bacillus subtilis, the general stress response is controlled mainly by ^B, the alternativetranscription factor of the RNA polymerase that brings abouta special physiological state which significantly enhances bacterialsurvival [11, 20, 22, 23, 29, 32, 33, 37] . It is estimated thatover 200 genes [5% of the coding capacity of the genome] aredirectly or indirectly under ^B control, and the loss of ^B functionleads to multiple-stress sensitivity, compromising the survivalof the ^B null mutant strain [23, 29, 32] . Besides having thisvery important, rapid, reversible, and plastic adaptive response[22, 29], B . subtilis is also able to differentiate into dormantspores when nutritional conditions become so extreme that the ^B-dependent response would not be adequate to guarantee thesurvival of the cell [19, 21, 24, 30, 31] . While ^B is the keyregulatory protein involved in the reversible adaptive stressresponse of vegetative cells, the master transcription factor Spo0A is the key regulator responsible for the decision of a vegetative cell to differentiate into a dormant and highly resistant new cell, i.e., the spore [31] . It is accepted that these responses,general stress adaptation and sporulation, are important forthe survival of B . subtilis in its natural environment, i.e.,soil [29-33] . Furthermore, high levels of expression of generalstress proteins provide stressed or starved cells with multiple,nonspecific, protective functions for future or unexpected insults[37] . In particular, soil is subject to important fluctuationsof the environmental temperature, which can vary from mesophyllicvalues at midday to chilling temperatures at night [28, 41].Taking these observations into consideration, we considered it to be of interest to analyze whether ^B and/or Spo0A mightplay a role in the stress adaptation and survival of B . subtilisduring growth and permanence at a low growth temperature.

 
Bacterial strains, media, and growth conditions. The experiments conducted in this study were performed withwild-type reference strain JH642 and isogenic derivatives [38]. Mutations and gene reporter fusions were introduced into strain JH642 by transformation of competent cells as previously described[3] . The parent strain and the resulting isogenic derivativesare described in Table 1 . Bacteria were routinely grown under vigorous agitation [220 rpm] in Spizizen minimal medium [MM], with 0.5% [wt/vol] glucose as the carbon and energy source and L-tryptophan [50 µg/ml] and L-phenylalanine [50 µg/ml][14] . Where indicated below, the strains were grown in Schaeffersporulation medium or Luria-Bertani [LB] medium . For sporulationefficiency, cells were grown in MM for the times indicated inTables 2 and 3 and the figure legends and then treated withCHCl₃ or heated at 80°C for 15 min before being plated [38]. For drug resistance selection in B . subtilis, antibiotics were used at the following final concentrations: 5 µg/ml for chloramphenicol, 1 µg/ml for erythromycin, 2 µg/mlfor kanamycin, and 25 µg/ml for spectinomycin.

 
Determination of ß-galactosidase activity. For the determination of the ß-galactosidase activityof the lacZ gene reporter fusions, cultures were propagatedas described in the figure legends . At appropriate times, triplicate1-ml aliquots were removed and harvested by centrifugation at4°C . ß-Galactosidase enzyme assays were conductedas described previously [3].

Western blot analysis. B . subtilis cultures of the parent strain JH642 and its isogenicderivate Sik31 [spo0A::Ery^r P_spac-spo0Asad67] [Table 1] were grown in MM and LB medium, respectively . Aliquots of 20 ml of each culture were collected by centrifugation and washed threetimes in disruption buffer {50 mM TES [N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid], 50 mM NaCl, 5 mM dithiothreitol, 10% glycerol, 1 mM EDTA [pH 7.5], and protease inhibitor cocktail [Complete; Boehringer]}. Cells were finally resuspended in 0.5 ml of disruption bufferand disrupted by sonication [six times, 10 s each time] usinga model 200 sonifier [Branson] . Cell debris was removed by centrifugation[10 min, 13,000 rpm at 4°C [Marathon 16 Km; Fisher Scientific]].Protein concentrations in crude extract were determined by usingthe Bradford protein assay [Bio-Rad] [8a] . The samples were subjected to sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis, transferred to an Immobilon membrane [Millipore, Bedford, Mass.], and revealed by using anti-Spo0A rabbit antibody and an alkaline phosphatase-conjugated anti-rabbit immunoglobulin G [IgG] [Bio-Rad]. For signal quantification, films were scanned and analyzed densitometrically.The Sik31 culture was grown in LB medium at 37°C until themid-exponential phase . At this point, IPTG [isopropyl-ß-D-thiogalactopyranoside; 1 mM] was added to half of this culture, and growth continued for another 2 h . After this induction period [production of Spo0A-Sad67], the culture was processed for protein analysis.

 
Expression of sigB and spo0A at a low growth temperature. It was previously shown that the measurement of ß-galactosidase activity from transcriptional lacZ fusions constitutes a satisfactory method to analyze the response of B . subtilis genes to cold shock [1, 2] . Therefore, we assayed the effect of the cold shock[from 37 to 20°C] on the expression of sigB [coding for ^B] and spo0A in isogenic B . subtilis strains harboring transcriptionallacZ fusions to the promoter regions of both regulatory genes[Table 1] . Figure 1A shows a typical growth curve of a cultureof B . subtilis grown in MM [Spizizen salts supplemented with0.5% glucose, 50 µg of Trp/ml, and 50 µg of Phe/ml]with strong aeration [220 rpm] until the early exponential phase[optical density at 525 nm [OD₅₂₅], 0.25], half of which wasthen transferred from 37 to 20°C . The cold-shocked culturedid not show a lag phase, and the generation times under theseconditions of exponential growth at 37 and 20°C were 2.5and 13.0 h, respectively [Fig . 1A] . Despite the important reductionin the rate of growth, the cold-shocked culture reached thesame cellular density as that of the culture that remained at37°C [Fig . 1A] and reached similar cellular yields at bothtemperatures [an average of 1.0 x 10⁸ to 4.0 x 10⁸ CFU/ml [datanot shown]] . Under these experimental conditions, comparingcultures maintained at 37°C, we observed a reproducible10-fold increase in sigB expression and a significant induction[four- to fivefold increase] of spo0A expression during thevegetative and stationary phases of the cultures transferredto 20°C [Fig. 1B and C] . Interestingly, despite the absenceof a lag phase, the cold induction of sigB and spo0A after the temperature downshift was not immediately perceptible . This delay in gene activation is intriguingly different from whatoccurs with the widely studied induction of sigB that followsthe input of metabolic or environmental stress signals at 37°C[7, 8, 23, 29, 33] or the rapid response of the cold shock regulon of B . subtilis [6, 16-18, 39] . Effectively, all these adaptiveresponses are fully displayed after the first 15 min followingthe stress [6, 29, 33] . By contrast, in our study, the inductionof sigB and spo0A began to be noticeable only 4 to 5 h followingthe temperature downshift but many hours before [40 h] the cold-shockedcultures reached the stationary phase of growth at 20°C[Fig . 1B and C] . Interestingly, this apparent delay of ^B inductionduring adaptation to the cold had been previously reported tooccur during the cold shock response of the psychrotrophic food-bornepathogen Listeria monocytogenes [5] . In this bacterium, the ^B-like transcription factor was induced by the cold shock [from37 to 8°C] only 4 h following the temperature downshift,with a substantial increase in sigB expression after 6 h ofthat treatment [5] . Furthermore, as is the case for sigB inB . subtilis, the induction of the ^B-like transcription factorof L . monocytogenes by other stresses [i.e., osmotic stress]at 37°C occurs during the 10 min that follows the stressingsignal [12] . Thus, we deduced that although sigB and spo0A arenot components of the rapidly induced cold shock regulon ofB . subtilis [6], they may constitute a second repertoire ofgenes induced during the vegetative growth of the adapting cellto the low growth temperature.

 
Since spo0A expression is driven from two promoters, a constitutive and weak ^A-dependent promoter during vegetative growth and astronger ^H-dependent promoter activated at the end of the exponentialgrowth [time zero [T₀]], we wondered on which sigma factor [^A or ^H] the observed induction of spo0A expression during the vegetative growth at 20°C depended . Additionally, the transcription factor ^H, encoded by spo0H, is not only essential for the burstof spo0A transcription that starts at the beginning of the stationary phase [T₀] at 37°C [4, 21, 24, 31] but also required for the survival of B . subtilis under extreme growth conditions [11] . Therefore, we first corroborated that the ^H-coding genespo0H was also expressed during the logarithmic phase of cold-shockedcells [Fig . 1D] . Then, we confirmed that the activity of ^H wasessential for the induction of spo0A transcription at 20°C since a spo0H deletion strain was completely unable to induce the spo0A-lacZ fusion after the temperature downshift [Fig. 1E] . We also show [Fig . 1] that the levels of ß-galactosidaseactivity that accumulated in ^H-deficient cells at both temperatures[37 and 20°C] did not significantly differ from one another.This result strongly indicates, as expected [1, 2], that theenhanced ß-galactosidase activity of the reporterfusions detected in wild-type cells after the temperature downshiftreflected a real induction of the cold-shocked genes and nota passive accumulation of ß-galactosidase as a consequenceof a conceivably greater stability of the enzyme at the lowtemperature . Moreover, the levels of ß-galactosidase activity accumulated by strain GS37 [Table 1], harboring a transcriptionallacZ fusion to the promoter region of the non-cold shock-induciblefabH gene [36], were essentially the same at 37 and 20°C[data not shown].

It has been extensively observed that the induction of sigB and spo0A expression in cultures of B . subtilis grown at 37°C under nonstress conditions occurs only at the beginning of the stationary phase of growth [T₀] . It is believed that this postexponentialinduction of sigB and spo0A expression at 37°C allows thegrowth-restricted cells to adapt [^B-dependent response] or sporulate[Spo0A-dependent response] under the unfavorable conditionsprevailing during the stationary phase [23-25, 29, 33] . By contrast,the present results indicate that sigB and spo0A expression is induced after a temperature downshift from 37 to 20°Cand suggest that ^B and Spo0A would be overproduced in cold-shockedcells of B . subtilis many hours before the beginning of thestationary phase of growth . Moreover, the unexpected high levelsof induction of sigB and spo0A expression during the logarithmicgrowth of B . subtilis at 20°C [Fig . 1B and C] leaves open the possibility that ^B and/or Spo0A plays a previously unrecognizedrole during the adaptive response of this bacterium to the cold[Fig . 1F].

Spo0A is overproduced and active during the vegetative phase of cold-shocked cells. As we have shown, the expression of the regulatory genes spo0Aand spo0H was highly induced after the temperature downshift[Fig . 1C and D] . Since the expression of both genes is controlledby a positive autoregulatory loop [3, 4, 24, 25, 31] that requireshigh levels of the active phosphorylated form of Spo0A [Spo0AP], it can be hypothesized that the observed upregulation of ß-galactosidase activity derived from the spo0H-lacZ and spo0A-lacZ fusionsat the low temperature reflected an overproduction of Spo0A during the vegetative phase . Hence, it was conceivable to hypothesize that the active form of Spo0A [Spo0AP] should be present inhigher levels at 20°C than at 37°C . Effectively, Westernblot experiments using anti-Spo0A antibodies [4] confirmed theoverproduction of Spo0A during the logarithmic growth of B.subtilis at 20°C [Fig . 2] . The vegetative levels of Spo0Aat 20°C were severalfold higher than the levels of the regulatoryprotein found in logarithmic cells of the same strain [JH642]grown at 37°C [Fig . 2] . This result confirmed the overproductionof Spo0A and strongly suggested that Spo0AP was predominantduring logarithmic growth at the low temperature . Effectively,this presumption was confirmed since the expression of two exclusiveSpo0AP-dependent sporulation genes [spoIIA and spoIIG], whichare normally activated after T₀ at 37°C, was dramatically induced many hours [40 h] before the cold-shocked culture reachedthe stationary phase of growth [T₀] [Fig . 3] . These results confirmed the cold induction of spo0A and the overproduction of active Spo0A [Spo0AP] during the vegetative growth of B.subtilis at the low temperature [Fig . 3C].

 
A loss of Spo0A, but not the loss of the sporulation ability, results in diminished survival and a higher sensitivity to stress for cold-shocked vegetative cells. The high levels of spo0A expression [Fig. 1C] and Spo0A production[Fig . 2] and the evidence for its activity [Fig . 3] in wild-type cells during exponential growth at 20°C prompted us to analyze a potential improvement of the sporulation ability at the low temperature . To this end, we determined the kinetics of spore formation during the growth of wild-type strain JH642 at 20and 37°C . As shown in Table 2, the wild-type strain formed low numbers of spores [fewer than 10⁵ spores/ml] during the logarithmic and the early stationary phase of growth that were almost identical at both growth temperatures . This result suggested that the high level of Spo0A production [Fig . 2] and its prematureactivity [Fig . 3] during the logarithmic phase of the culturesgrown at 20°C were not related to an improvement of thecapacity to make spores at the low temperature . Furthermore,after 2 or 3 days in the stationary phase, the increase in thenumber of mature spores was more marked for the culture maintainedat 37°C [an average of 5 x 10⁷ spores/ml] than for the culturemaintained at 20°C [1 x 10⁶ spores/ml] [data not shown].The fact that spo0A was highly expressed at 20°C many hours[70 to 80 h] before a significant number of spores were formed[Table 2] suggested that the activity of the transcription factor was required for a role other than that of premature spore formation. These results induced us to hypothesize that Spo0A plays a role in the survival and/or the development of full stress resistanceof cells that were shocked by a temperature downshift . To testthis hypothesis of a non-sporulation-related role of Spo0A duringthe adaptation of vegetative cells to the cold, we first examinedthe growth features and the survival properties of the reference wild-type strain JH642 and its isogenic spo0A mutant strain JH646 [Table 1] . The growth of both isogenic strains did not show any significant difference among the lag phases, generation times, and the final cellular yields in MM at 20°C [datanot shown] . However, after the commencement of the stationaryphase at 20°C, a reduction of the OD of the spo0A mutantwas evident compared with that of the wild-type strain, whichsuggested that the mutant was dying faster than its parentalstrain [Fig . 4A] . To determine the cellular viability duringthe stationary phase, we plated serial dilutions of both strainsafter their permanence during 7 days in the stationary phaseat the low temperature . The result was a reproducible 10,000-foldloss of viability of the spo0A mutant relative to that of thewild type [data not shown] . In fact, after a week at the lowtemperature, the average counts of viable cells from five independentexperiments were 3 x 10³ CFU/ml for the spo0A mutant and 3 x10⁷ CFU/ml for the wild-type strain . To address the possibilitythat this dramatic loss of viability was due to the impairedsporulation ability of the spo0A mutant, we tested the viabilityof a sigF null mutant strain grown at 20°C [3]. ^F, the productencoded by sigF, is the first sporulation-specific sigma factorthat becomes active during sporulation, and the loss of itsfunction completely blocks spore formation but does not impair Spo0A synthesis and its activation by the phosphorelay signaling system [3, 24, 31] . Figure 4A shows that the survival of thesigF null mutant was indistinguishable from that of the wildtype after a prolonged incubation at the low growth temperature.After 3 weeks in the stationary phase at 20°C, the viablecounts for both sporulation-deficient strains, the sigF andspo0A mutants, were 5 x 10⁶ and 2 x 10² CFU/ml, respectively. Since the only difference between both asporogenous strainswas the ability to produce Spo0AP, it can be concluded thatthe diminished viability of the spo0A mutant strain at the lowtemperature was specifically due to the loss of Spo0A functionand not to a general loss of sporulation ability . In this regard,one of the first functions of active Spo0A is that of repression[achieved by very low levels of Spo0AP] of abrB [3, 24, 25, 31] . AbrB is a key transcription factor responsible, as longas the provision of nutrients is adequate to maintain the vegetativegrowth, for the repression, direct or indirect, of many stationary-phasegenes [24, 25, 31] . Since the AbrB levels fall as the Spo0AP levels increase [3, 31], the possibility existed that the requirementof Spo0AP for the survival of nonsporulating cells during thestationary phase at a low temperature is indirect due to theupregulated levels of AbrB in the spo0A mutant strain [31].In this scenario, AbrB should play a negative role in the prolonged survival of cold-shocked cells instead of Spo0A playing a direct positive role in it . To test this possibility, we studied the survival properties of the wild-type strain and the spo0A mutant strain after the introduction of a null abrB mutation into both strains [Table 1] . As shown in Fig . 4A, the introduction ofthe abrB mutation did not alter the survival properties of eitherstrain . This result indicated that Spo0AP plays a novel anddirect role, apart from sporulation, in the adaptation to thecold and the survival of nonsporulating cells of B . subtilis[Fig . 4B] . In this respect, one important stress factor thatB . subtilis cells should frequently confront in the soil, itsnatural habitat, is the fluctuation of environmental temperature[16-18, 28, 39, 41] . Therefore, we tested the ability of B.subtilis [the wild type and the spo0A and sigF mutant strains]grown at 20°C until the beginning of the stationary phase[T₀] to survive the treatment at 54°C for 1 h [heat shock]before being plated . As shown in Table 3, the survival ratesof the Spo⁺ wild-type strain and the Spo^- sigF strain were essentially the same . By contrast, the spo0A mutant strain showed a 1,000-fold decrease in the percentage of cells that survived after the heat shock treatment . This result indicated that the inabilityto synthesize Spo0A, and not the inability to sporulate, wasthe cause for the higher sensitivity of the cold-shocked cellsto heat shock . This result strongly reinforces the hypothesisof a new and important role for Spo0AP in the development offull stress resistance and the survival of cold-adapted cellsunder nonsporulation conditions [Fig. 4B].

 
A loss of ^B function results in a decreased rate of survival of cells grown at a low temperature. Since ^B activity is posttranslationally controlled by a cascadeof regulatory proteins [32], we wondered whether the sigB induction that followed the temperature downshift [Fig . 1B] reflectedan upregulated activity of ^B during the logarithmic phase ofthe cold-shocked cultures . To check this possibility, we measuredthe activity of the ^B-dependent fusion ctc-lacZ, which is atraditional reporter of ^B activity [7, 8, 12] . The expressionof ctc was also dramatically induced after the cold shock [Fig.5A], suggesting that the alternative transcription factor ^B was active during the logarithmic growth of B . subtilis at 20°C. To test the physiological role of ^B during growth at the lowtemperature, we compared the levels of growth of wild-type strainJH642 and its isogenic sigB mutant strain MR644 at 20°C[Table 1] . Comparison of the growth patterns of sigB mutantcells and wild-type cells after the temperature downshift revealedno difference between the two strains [JH642 and MR644] [datanot shown] . However, as was the case with the spo0A mutant,the ^B-deficient cells started to lyse soon after the commencementof the stationary phase [Fig . 5B] . The average viable-cell counts after a week at 20°C were 5 x 10⁷ CFU/ml for the wild-typeand 1 x 10⁶ CFU/ml for the sigB mutant strain . This moderate but reproducible requirement of ^B activity for survival duringcold temperatures was observed previously when B . subtilis confrontedother environmental or metabolic stresses [23, 32, 37] . Thus,the present results enlarge the horizon of the known scenariosthat require the activity of ^B for a better adaptation of B.subtilis after an environmental insult [Fig . 5C].

 
A loss of ^B function results in a delayed and decreased sporulation proficiency for B . subtilis at a low growth temperature. Interestingly, the sigB mutant strain, apart from its decreasedviability at 20°C, showed a clear oligosporic phenotypeafter the growth on sporulation plates during a week at 20°C[Fig . 6A] . This result prompted us to further examine whether ^B was required for survival and/or sporulation of the cold-shocked cells . Therefore, we monitored the cellular viability and the sporulation proficiency of wild-type strain JH642 and its isogenic sigB mutant derivate MR644 [Table 1] at 20°C . The cold-shockedsigB⁺ and sigB mutant cultures, having similar generation times[data not shown], reached essentially the same number of viablecells at the beginning of the stationary phase [T₀] [4 x 10⁸ CFU/ml for the ^B-proficient cultures and 5 x 10⁸ CFU/ml for the ^B-deficient cultures] [Fig . 6B] . However, soon after T₀, i.e., 1 to 18 h after T₀ [T₁ to T₁₈], there was a reproducible10- to 15-fold loss of cellular viability for the sigB mutantculture [Fig . 6B] . As shown earlier [Fig . 5B], this moderatebut reproducible loss of cellular viability had previously beenobserved in ^B-deficient cells after different challenges [7, 8, 11, 23, 37] and confirms that the loss of ^B function hasa modest but significant effect on cellular viability at thelow temperature . In fact, ^B-deficient cells challenged at thebeginning of the stationary phase [T₀] by heat shock [54°C,1 h] showed a survival percentage of 0.5 [data not shown] . Thisvalue was between the survival rates obtained for the wild-type[3.3%] and the spo0A mutant [0.002%] strains after the heattreatment [Table 3] . Remarkably, the ^B-deficient culture showeda lower capacity to make spores than the culture proficientin ^B production [Fig . 6B] . Throughout the stationary phase at 20°C, which lasted for 3 weeks, and even before or at T₀, when the numbers of viable cells were similar for the two strains, the number of spores formed by the sigB mutant culture was always significantly lower than the number of spores obtained withthe sigB⁺ counterpart [Fig . 6B] [T_-0.5 to T₀] . Finally, after20 days in the stationary phase, both cultures [sigB⁺ strainand sigB mutant] were completely sporulated, with a 50-foldloss of spore efficiency for the ^B-deficient culture . This significantand reproducible oligosporic phenotype of the ^B-deficient culturesuggested that the activity of ^B was necessary, apart from itsrole in stress survival of vegetative cells, for the completeproficiency in the sporulation of B . subtilis grown at a lowtemperature [Fig . 6A].

 
The delay in spore formation and the reduced sporulation yield observed for the sigB mutant cultures at 20°C [Fig . 6] promptedus to better understand the novel requirement of the ^B activityfor efficient sporulation at a low growth temperature . To determinethe stage at which sporulation was delayed and/or affected bythe absence of ^B activity at 20°C, we measured the expressionof several sporulation genes that are under temporal and spatialregulation during the normal development of the spore [Fig.7] [31] . Despite the observed delay and the reduced efficiencyin spore formation of the ^B-deficient cultures at 20°C [Fig.6], the ß-galactosidase activities accumulated fromthe Spo0AP-, ^F-, ^E-, and ^G-dependent genes [Fig . 7A to D] wereremarkably higher for the sigB mutant cultures than for thesigB⁺ parental strains . In contrast, the ß-galactosidaseactivities accumulated from the same strains grown at 37°Cwere indistinguishable for the sigB⁺ and sigB mutant cultures[data not shown] . The upregulated accumulation of ß-galactosidaseactivity under Spo0AP, ^F, ^E, and ^G control in sigB mutant cellsat 20°C [Fig . 7A to D] plus the oligosporic phenotype ofthese cultures at the low temperature [Fig . 6A] strongly suggestedthat ^B was required at a late stage of the morphogenesis ofthe spore beyond ^G activation [31] . In this respect, it is interesting to mention that in the same ^B-dependent ctc operon there isa gene called spoVC [29, 33, 42] . A thermosensitive spoVC mutantstrain ceases sporulation at a late stage of development with a deficient spore cortex development and coat formation, both of which are under ^K control [29, 42] . Since spoVC is underthe control of ^B activity [29, 33], it is tempting to speculatethat the poor sporulation of sigB mutant cells at 20°C isa consequence, direct or indirect, of the poor or null activationof the ctc and spoVC genes in the absence of ^B activity [Fig.7E].

 
sigB induction is interconnected with the Spo0AP activity in B . subtilis. The dramatic induction of sigB and spo0A after the temperaturedownshift [Fig . 1B and C] led us to examine whether there wasa connection between the expressions of both regulatory genes[Fig . 8A] . Since ^B is required for the expression of its codinggene sigB [29, 33] and the same requirement is valid for Spo0AP and the induction of spo0A [3, 31], we used sigB-lacZ and spo0A-lacZfusions to monitor whether ^B and/or Spo0AP is essential forthe cold induction of spo0A and/or sigB, respectively [Fig.8A] . The expression of the Spo0AP-^H-dependent spo0A-lacZ fusionwas induced, as expected, at 20°C, and this induction wasnot affected by the absence of ^B activity [Fig . 8B] . In contrast,even though the induction of sigB was not affected by the presenceor absence of Spo0AP, there was a significant enhancement ofsigB expression in a spo0A mutant background at both temperatures[20 and 37°C] [Fig. 8C] . These results indicated that thecold induction of sigB and that of spo0A were independent from one another [Fig . 8A, diagram III] . In addition, the higheractivity of sigB in the spo0A mutant strain [Fig . 8C] suggestedthat Spo0AP has a direct or indirect detrimental effect on thelevels of expression of sigB [Fig . 8D] . Perhaps it is conceivable to hypothesize that in the absence of Spo0AP activity, the alternativetranscription factor ^B might be upregulated to ameliorate theeffects of the absence of Spo0AP activity in spo0A mutant cellsunder stress conditions [Fig. 8D].

 
Free-living bacteria have the capacity to react rapidly to sudden upshift and downshift changes of the growth temperature [2, 6, 13, 16-18, 28, 41] . In particular, B . subtilis has becomean attractive model for study not only because of its capacityto undergo cellular differentiation [31] but also because ofour ability to study the bacterium's cold shock response [2,6, 13, 16-18, 28, 41] . In this particular case, B . subtilis responds to a decreasing temperature with a rapid inductionof a little less than 100 genes that conform to the cold shockregulon [6] . The immediate result of this cold induction isa characteristic strong repression of major cellular metabolicactivities, whereas only a limited number of processes essentialfor cold adaptation [principally translation machinery and membraneadaptation] are induced [2, 6, 13, 16-18, 28, 41] . Among therelevant members of this regulon whose expression is inducedby cold, there is a two-component system [desK-desR] that hasrecently been reported to regulate the expression of anothercold-induced gene [des] that codes for an acyl-lipid desaturaseenzyme [1, 2, 6, 10] . With decreasing temperature, the membrane-boundsensor histidine kinase DesK phosphorylates its correspondingresponse regulator, DesR, which then binds to a specific recognitionsequence in the promoter region of the ⁵ desaturase-coding genedes to activate its transcription [1, 2] . The activity of thefatty acid desaturase Des, located in the membrane, finallymaintains the fluidity of the membrane in the cold [1, 2, 10,13, 14, 40] . Additionally, the expression of the genes codingfor the DNA gyrase [gyrA and gyrB, which increase the negative supercoiling of the DNA] and the DNA topoisomerase I [topA, which relaxes the negative supercoiling] is induced and repressed, respectively, after the cold shock [6] . This change in the balanceof the DNA-modifying enzymes seems to be at least a part of the system regulating membrane fluidity since it has been previously demonstrated that an increment in the negative DNA supercoiling of B . subtilis was essential for the transcriptional induction of des after cold shock [15, 26].

The main contribution of the present work is the evidence thatthe very recently characterized cold shock response [6] is not sufficient for the development of a complete adaptation to low temperatures, and hence of the survival, of B . subtilis . In fact, the present study demonstrates that the genes encodingthe key transcription factors Spo0A and ^B are strongly inducedafter a temperature downshift from 37 to 20°C [Fig . 1].This transcriptional induction of spo0A and sigB was not immediate,as it was for the genes belonging to the cold shock regulonthat were induced during the first minutes following the temperaturedownshift [6], but occurred during logarithmic growth and manyhours [45 h] before the cold-shocked cultures reached the stationaryphase of growth [Fig . 1] . This delay in gene activation might explain why the cold induction of sigB and spo0A was not observedin previous proteome and transcriptome studies that analyzed the initial response of B . subtilis to cold shock [6, 16-18,39] . Our results suggest that spo0A and sigB might correspond, as would be the case for the sigB-like gene of L . monocytogenes [5], to a second class of cold-induced genes whose functionis not related to an immediate adaptation of the cell to coldshock but is related to cellular viability and stress survival during long permanence at the low temperature.

It can be unquestionably asseverated that Spo0A is essentialfor spore formation at any temperature, but this work has clearly demonstrated that Spo0A has a novel sporulation-independentactivity required for the efficient survival of nonsporulatingresting cells at a low temperature [Fig . 4] . Furthermore, the strong spo0A expression during the logarithmic phase at 20°C [Fig . 1C] and the overproduction of active Spo0A [Fig. 2 and3] were not correlated with an improved ability of the cold-adaptedcells to form spores at the low temperature [Table 2] . Effectively,the number of spores formed at 20°C did not differ fromthe number of spores formed at 37°C during either the exponentialphase or the early stationary phase of growth [Table 2] . Thisresult opened the possibility that the overproduction of Spo0AP during logarithmic growth at 20°C might be required forthe development of full stress resistance before the cold-adaptedcells abandon exponential growth . In fact, cold-adapted cellsproficient in Spo0A activity at the end of exponential growth[T₀] were >2,000-fold more resistant to heat treatment [54°C,1 h] than were the equivalent cells deficient in Spo0A production[Table 3] . These properties were completely dependent on Spo0A activity and not on the sporulation ability or the activityof the transition state regulator AbrB [Fig . 4A] . Therefore, these results indicated a novel and important role for Spo0Ain cellular viability and stress survival at a low growth temperature [Fig . 9] . This new role of Spo0A might contribute to the preparationof B . subtilis cells for future stresses during long-term survivalas nongrowing vegetative cells in natural environments [Fig.4B and 9].

 
On the other hand, ^B was also dramatically induced and was requiredfor cellular survival during the stationary phase of cold-adaptedB . subtilis cells [Fig . 5] . Interestingly, during the reviewprocess of this work, Brigulla et al . reported the chill inductionof sigB expression in B . subtilis [9] . Using a proteome approachin conjunction with Western blot analysis and the measurementof ß-galactosidase activity from ^B-dependent reportergene fusions, those researchers showed the expression of the ^B regulon during continuous growth at a low temperature [9]. Those researchers showed that the growth of a sigB mutant strain was drastically impaired at 15°C but not at 20°C [9]. However, the survival and stationary-phase properties of ^B-deficient cold-shocked cells were not analyzed [9] . Effectively, in thepresent work we show the survival properties and sporulation phenotype of a sigB mutant strain grown and maintained at 20°C. ^B-deficient cultures started to lyse rapidly after the commencementof the stationary phase and were more sensitive to heat shockthan were ^B-proficient cultures at 20°C [Fig . 5 and datanot shown] . The rapid decline in the OD of the ^B-deficient culturesin the stationary phase at 20°C [Fig . 5B] was accompaniedby a delay and a rate of spore formation lower than that seenwith those cultures proficient in ^B production [Fig . 6] . Eventhough the sigB⁺ and sigB mutant cultures grown at 20°Cfinally reached 100% sporulation, the numbers of spores formedby the ^B-deficient cultures were 20- to 50-fold lower than thenumbers produced by the ^B-proficient cultures [Fig . 6B] . Theseresults reinforce the view that ^B is required for cellular viabilityat a low growth temperature and suggest a novel role of thiskey transcription factor in efficient sporulation at a low growthtemperature [Fig . 9] . In fact, the moderate but clear negativeeffect on efficient sporulation of the absence of ^B [Fig . 6]and the probable exigency of its activity at a late stage ofspore development at a low temperature [Fig. 7] were novel andunexpected . As mentioned previously, one gene under ^B controlpreviously reported to affect spore cortex and coat formation is spoVC [42] . Recently, it was demonstrated that its product,the protein SpoVC, has peptidyl-tRNA hydrolase activity thatis essential for vegetative growth and sporulation [27] . Peptidyl-tRNAhydrolase activity is essential for recycling tRNA moleculessequestered as peptidyl-tRNA as a result of premature dissociationfrom the ribosome during translation [27] . In nondividing cellsor during the last stages of spore development, in the absenceof new tRNA synthesis, the recycling by SpoVC of tRNA sequesteredas peptydil-tRNA would acquire high priority . A poor or nullactivation of the ctc-spoVC operon [29, 33] after a temperaturedownshift in sigB cells might be expected from the present resultsand might explain the observed oligosporic phenotype under theseconditions [Fig. 7E] . In addition, it is interesting to mentionthat Scott and coworkers have reported that Obg, an essentialGTP binding protein required for B . subtilis sporulation, wasassociated with the ribosome fraction and was necessary forthe stress activation of ^B [35, 36] . These observations point to the translational machinery and its putative associated proteins [Obg, SpoVC, and Ctc] for a possible coupling between sporulation and ^B-dependent stress adaptation [35, 36, 43] . In fact, wehave shown that in the absence of Spo0A activity, B . subtilisis unable to sporulate or fully adapt and that there is a significantimprovement in sigB expression [Fig . 8C] . Taking these resultsinto account, we suggest that sporulation and ^B-dependent stressadaptation are interconnected pathways that allow an adequate response of B . subtilis to its fluctuating environment [Fig. 9].

 
We are indebted to A . Grossman, W . Haldenwang, F . Kawamura,T . Leighton, R . Losick, P . Piggot, C . Price, P . Setlow, G . Shujman,W . Schumann, G . Spiegelman, and P . Zuber for providing strainsand suggestions during the course of the work . We also thankF . Kawamura and M . Fujita for the anti-Spo0A antibodies.

This research was supported by grants from the following national agencies: Fundación Antorchas [RG-14022-57], FONCyT [RG-PICT-0103379], and CONICET [RG-PIP-03052].

 
* Corresponding author . Mailing address: Facultad de Ciencias Bioquímicas y Farmacéuticas, Departamento de Microbiología, Subsuelo de Sala 9, Suipacha 531, Rosario-2000, Argentina . Phone: [54] 341-4353377 . Fax [54] 341-4804601 . E-mail: rrgrau@infovia.com.ar .

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