BACKGROUND
in nature, a key element of the adaptive responses of bacteria is the ability to sense and respond to the local environment, such as nutritional limitation, their population, the presence of toxic chemicals from other bacteria and host signals.
hence, it is important to coordinate the pattern of gene expression, and bacteria have evolved specific mechanisms to ensure the survival of the species in environmental niches.
for example, many bacteria use a variety of intercellular signaling systems including quorum sensing.
the intercellular signal molecules include n-acyl-homoserine lactones  in gram-negative bacteria, autoinducer  <dig>  and indole in both gram-negative and gram-positive bacteria, signal peptides in gram-positive bacteria, and others; these have been seen to co-ordinate gene expression for bioluminescence, sporulation, plasmid conjugal transfer, competence, virulence factor production, antibiotic production, and biofilm formation  <cit> .
indole is an intercellular signal  <cit>  as well as an interspecies signal  <cit> .
a variety of both gram-positive and gram-negative bacteria   <cit>  produce indole using tryptophanase  that can reversibly convert tryptophan into indole, pyruvate, and ammonia according to reaction below  <cit> .
indole plays diverse biological roles in the microbial community; for example, indole controls the virulence  <cit> , biofilm formation  <cit> , acid resistance  <cit> , and drug resistance  <cit>  in gram-negative bacteria.
in a gram-positive stigmatella aurantiaca, indole increases its sporulation via indole binding pyruvate kinase  <cit> .
moreover, recent studies suggest that abundant bacterial indole in human intestines plays beneficial roles in the human immune system  <cit> .
also importantly, indole increases escherichia coli antibiotic resistance, which eventually leads to population-wide resistance  <cit> .
p. alvei  belongs to the class bacillales, which includes bacillus, listeria, and staphylococcus and is an endospore-forming gram-positive bacterium that swarms on routine culture medium.
p. alvei is frequently present in cases of european foulbrood   <cit>  and has, on occasion, been the cause of human infections  <cit> .
p. alvei is the only indole-producing bacterium among many bacillus species  <cit> , and the biosynthesis of indole has been well-studied in p. alvei  <cit> .
it has long been thought that indole producing bacteria including p. alvei utilize tryptophanase to synthesize tryptophan and other amino acids from indole as a carbon source  <cit> .
however, the equilibrium of the reaction favors the production of indole from tryptophan  <cit> .
hence, we sought here the real biological role of indole in p. alvei physiology.
spore-forming bacteria can respond to nutritional limitation and harsh environmental conditions by forming endospores that are remarkably resistant to heat, desiccation, and various chemicals  <cit> .
spore formation is an elaborate and energy intensive process that requires several hours to complete  <cit> .
therefore, sporulation is a last-resort adaptive process that is tightly regulated by complex cell-cell signaling or so-called quorum sensing  <cit> .
bacillus subtilis produces multiple cell-cell signaling molecules to control the sophisticated sporulation  <cit>  that is often a temporal, spatial, and dynamic decision-making process  <cit> .
the outermost protective layers of b. subtilis endospores are the coat and the cortex  <cit> .
the spore coat is a barrier against bactericidal enzymes and destructive chemicals.
therefore, heat resistant spores are also resistant to treatment by various chemicals, such as acids, bases, oxidizing agents, alkylating agents, aldehydes and organic solvents  <cit> .
thus, we investigated the role of indole on heat resistance as well as other environmental stresses.
in this study, we identified that indole was a stationary phase extracellular molecule in p. alvei and functioned to inhibit spore maturation and to decrease survival rates under several environmental stresses.
additionally, we studied the effect of indole derivatives originated from plants on spore formation in p. alvei.
this study provides another important role of indole and indole derivatives.
RESULTS
extracellular indole accumulation in p. alvei
to be an environmental signal molecule, indole has to be excreted out of cells.
thus, the cell growth of p. alvei and the extracellular indole concentration were measured in luria-bertani  medium.
clearly, the level of extracellular indole from p. alvei was growth-dependent .
indole production was begun in the middle of exponential growth phase and reached the maximum amount  in the stationary phase.
notably, the level of extracellular indole present was stable over time at 37°c , which was one of characteristics of the indole molecule  <cit>  while other signaling molecules, such as ahls, ai- <dig>  and signal peptides, are only temporally present and heat-unstable  <cit> .
the accumulation pattern of extracellular indole was similar to that of other bacteria, such as e. coli  <cit>  and vibrio cholera  <cit> , while these two bacteria accumulated up to 500- <dig> μm of extracellular indole within  <dig> h in lb  <cit> .
the slower accumulation of indole in p. alvei was probably due to the 200-fold lower activity of p. alvei tryptophanase than that of e. coli tryptophanase  <cit> .
catabolite repression of p. alvei tryptophanase
since indole production was suppressed by the presence of glucose in e. coli through catabolite repression  <cit> , the effect of carbon sources on indole production was investigated in p. alvei.
similar to e. coli, the addition of glucose and glycerol  in lb medium completely abolished the production of indole in p. alvei for  <dig> h, while lactose  did not affect indole accumulation .
this result suggested that the indole accumulation in p. alvei was strictly controlled by catabolic repression although transport mechanisms of glucose and glycerol would be different.
in other words, p. alvei did not produce indole in the presence of the preferred carbon sources such as glucose and glycerol.
unlike the current observation, it was previously reported that the tryptophanase in b. alvei  appeared to be constitutive, and catabolite repression was not operative  <cit> .
the report studied the effect of only tryptophan on tryptophanase activity and found that the activity of p. alvei tryptophanase was independent of tryptophan  <cit> .
indole inhibits the heat-resistant cell numbers of p. alvei
the main hypothesis of this study was that a large quantity of extracellular indole would play a quorum sensing role in cell physiology of p. alvei so we investigated the effect of indole on sporulation and biofilm formation which was influenced by cell population and environmental stresses in other bacillus strains  <cit> .
in p. alvei, the addition of exogenous indole  surprisingly decreases the heat-resistant colony-forming unit  in a dose dependent manner .
for example, indole  decreased the heat-resistant cfu of p. alvei compared to no addition of indole 51-fold at  <dig> hr  and 10-fold at  <dig> hr .
to confirm the presence of exogenous indole, the indole level in dsm medium was measured with hplc.
the level of exogenous indole  was not changed at all over  <dig> h .
hence, the exogenous indole was not utilized as a carbon source and inhibited the heat-resistant cfu of p. alvei.
additionally, the temperature effect of indole on the heat resistance of p. alvei was investigated since the environmental temperature affected indole signaling in e. coli  <cit> .
unlike in e. coli, the inhibitory effect of indole  on the heat-resistant cfu of p. alvei at 30°c  was similar to that at 37°c in p. alvei .
hence, it appeared that the temperature effect of indole on the heat-resistant cfu of p. alvei was not significant under the tested laboratory conditions.
indole inhibits the development of spore coat and cortex
the effect of indole on the morphology of sporulating cells was examined by transmission electron microscopy.
surprisingly, the proportion of sporulating cells in the total number of cells was similar between with and without treatment of indole .
however, exogenous addition of indole influenced the morphology of the spore coat and the cortex.
cells with exogenous indole formed endospores with a thin spore coat and a thin spore cortex, while using no indole treatment resulted in a thick spore coat and cortex .
because the spore coat and cortex were important for heat resistance and chemical resistance  <cit> , we concluded that indole caused an immature spore that negatively contributed to the heat resistance of p. alvei.
effect of indole derivatives on the heat resistance of p. alvei
in the natural environment, indole can be easily oxidized into hydroxyindoles by diverse oxygenases, and indole derivatives often show different effects on bacterial physiology  <cit> .
thus, p. alvei can often encounter many kinds of indole-like compounds that are synthesized from tryptophan in other bacteria, plants, and even animals.
therefore, seven indole derivatives have been further investigated for the heat resistance of p. alvei.
as a negative control, glucose was used since glucose decreased the sporulation of b. subtilis  <cit> .
similar to b. subtilis, glucose  clearly decreased the heat-resistant cfu by 600-fold in p. alvei .
however, l-tryptophan as the main substrate of the indole biosynthesis did not have much influence on the heat-resistant cfu, which supported that indole rather than tryptophan specifically influenced the heat resistance of p. alvei .
most interestingly, a plant auxin, 3-indolylacetonitrile dramatically  decreased the heat-resistant cfu of p. alvei in a dose dependent manner at  <dig> and  <dig> hr , while another auxin 3-indoleacetic acid had a less significant influence, and tryptamine and 2-oxindole had no effect .
therefore, these results suggest that the functional groups of indole derivatives may control the development of p. alvei spores.
similar to indole, the proportion of sporulating cells in the total number of cells was similar with and without treatment of 3-indolylacetonitrile .
also, 3-indolylacetonitrile produced an irregular spore coat, while no treatment produced sturdy coat .
therefore, it appeared that indole and 3-indolylacetonitrile inhibited spore maturation rather than sporulation initiation.
in order to understand how most spores  in the presence of indole and 3-indolylacetonitrile could not survive against heat treatment, the lysozyme resistance assay  <cit>  was performed with 30-hour grown cells since the lysozyme treatment could release all spores.
as a result, indole and 3-indolylacetonitrile produced a large portion of lysozyme-resistant cells  which are probably the number of total spores, while indole and 3-indolylacetonitrile produced only  <dig>  ±  <dig> % and  <dig>  ±  <dig> % heat-resistant cells ; hence it appeared that a large number of spores have some spore defect for heat resistance.
therefore, it appeared that the low heat-resistant cfu was caused by some spore defect or the altered spore structure.
furthermore, the effect of indole and 3-indolylacetonitrile was investigated using another spore-forming medium, brain heart infusion  agar for a longer incubation time  when sporulation process would be completed.
similar to dsm medium, indole  and 3-indolylacetonitrile  inhibited the heat-resistant cfu of p. alvei , compared to no addition of exogenous indole .
therefore, the inhibitory impact of indole and 3-indolylacetonitrile was effective in different media for a long term, while their effect on heat resistance was attenuated with a longer incubation time.
effect of indole and indole derivatives on cell growth
to test the toxicity of indole and indole derivatives, cell turbidity at  <dig> hr and the specific growth rates with indole and 3-indolylacetonitrile were measured.
most indole derivatives at the concentration tested  did not have much of an inhibition effect on the cell growth of p. alvei, while indole-3-acetamide and 2-oxindole  slightly decreased cell growth .
the growth rate of p. alvei was  <dig>  ±  <dig> /h in the absence of the indole derivatives in lb medium, whereas the growth rate was  <dig>  ±  <dig> /h with indole  and  <dig>  ±  <dig> /h with 3-indolylacetonitrile .
in dsm medium, the growth rate of p. alvei was  <dig>  ±  <dig> /h in the absence of the indole derivatives, whereas the growth rate was  <dig>  ±  <dig> /h with indole  and  <dig>  ±  <dig> /h with 3-indolylacetonitrile .
therefore, indole and 3-indolylacetonitrile were not toxic to p. alvei and the inhibitory effect of the heat resistance was mostly due to the function of indole and 3-indolylacetonitrile rather than growth inhibition.
indole contributes to low survival against environmental stresses
since endospores are remarkably resistant to heat as well as various chemicals  <cit> , we presumed that indole also decreased the resistance to environmental stresses, such as treatment with antibiotics, ethanol and low ph.
as expected, indole decreased the survival rates with three antibiotics  and when exposed to low ph and 70% ethanol .
for example, indole decreased tetracycline resistance  <dig> -fold, erythromycin resistance  <dig> -fold, and chloramphenicol resistance 4-fold, and the survival rates with ethanol  <dig> -fold and ph  <dig>  21-fold, respectively.
these results are a good match with the sporulation results .
effect of indole on the survival of b. subtilis spores
since p. alvei belongs to the same bacillales order including b. subtilis , the effect of indole and 3-indolylacetonitrile was investigated in b. subtilis that did not produce indole .
unlike p. alvei, indole and 3-indolylacetonitrile had no impact on the heat resistance in b. subtilis, while glucose treatment as a negative control significantly decreased the heat-resistant cfu .
hence, it appeared that the action mechanism of indole was different between indole-producing p. alvei and non-indole-producing b. subtilis.
discussion
indole is an abundant environmental signal in both gram-positive and gram-negative bacteria  <cit> .
currently, the diverse roles of indole as an intercellular signal are beginning to be revealed in various indole-producing-bacteria, such as e. coli  <cit> , vibrio cholerae  <cit> , stigmatella aurantiaca  <cit> , fusobacterium nuceatum  <cit> , and porphyromonas gingivalis  <cit> , as well as in non-indole-producing bacteria, such as pseudomonas aeruginosa  <cit>  and salmonella enterica  <cit> .
the current study shows that the environmental signal indole also has a role in gram-positive p. alvei.
interestingly, the role of indole seems to be substantially divergent in different microorganisms, reflecting adaptation to different environments and niche-specific challenges.
for example, indole differently controls  biofilm formation in different e. coli strains  <cit> , vibrio cholerae  <cit> , and fusobacterium nuceatum  <cit> .
also, indole and indole derivatives induced sporulation in stigmatella aurantiaca  <cit> , while this study shows that indole reduced the integrity of spores in p. alvei .
therefore, the results suggest that different bacterial species have developed their unique systems to beneficially utilize indole in their microbial community.
previously, it was reported that indole derivatives, such as 3-indoleacetic acid, 3-indolylacetonitrile, tryptamine, and 2-oxindole, but not indole, decreased the percentages of spore germination and appressorium formation, which inhibited all stages of infection behaviors in a rice pathogen magnaporthe grisea  <cit> .
these results and the current study suggest that indole derivatives, such as 3-indolylacetonitrile, can be used as protective compounds against spore-forming p. alvei.
since indole influenced the biofilm formation of several indole-producing bacteria, such as e. coli  <cit> , vibrio cholerae  <cit> , and fusobacterium nuceatum  <cit> , and the sporulation transcription factor spoa was required for biofilm development in b. subtilis  <cit> , the effect of indole on the biofilm formation of p. alvei was investigated.
however, indole did not show an effect on p. alvei biofilm formation in the 96-well plate biofilm assay in lb or dsm media either at 30°c and at 37°c .
therefore, the indole-involving mechanism of p. alvei biofilm formation is different from that in other strains.
glucose obviously prevented the development of cfu of p. alvei presumably by preventing sporulation  as well as in b. subtilis via catabolite repression  <cit> .
because indole is a stationary phase signal  and because the production of indole is tightly regulated by carbon sources , the role of indole on spore formation could be closely affected by the catabolite repression.
thus, indole serves not only as an indicator of cell population, but also as an indicator of starvation.
this dual function of indole may reflect the status of cells in the environment.
because the accumulation of extracellular indole can be dramatically affected by many environmental factors  in addition to carbon sources  <cit> , the action of indole would be governed by the environment in a sophisticated manner.
nevertheless, the question remains as to why p. alvei produces copious amount of extracellular indole, as it causes immature spore formation .
one possible explanation can be found in the previous study in that bacteria utilize indole as a defense tool against non-indole producing pathogenic p. aeruginosa to diminish its virulence  <cit> .
another possible answer is that indole intentionally lowers integrity of spores in order to make cells easy to resume growth when the environment is favorable again at a later date.
hence, a large quantity of indole is an indicator of a favorable environment in which other unfavorable species are scare and indole may control the timing of germination in natural environments.
although highly speculative, another possibility is that indole signal negatively controls spore maturation, while other quorum sensing molecules positively regulates sporulation of bacillus, even using multiple signaling molecules  <cit> .
also, there is the possibility that indole is affecting spore germination since indole lowered the survival against environmental stresses  while the number of spore was not affected by indole .
however, it is unclear, so far, how the indole signal influences sporulation in p. alvei.
it is necessary to identify the operon of p. alvei tryptophanase to understand the genetic regulation of indole biosynthesis.
for further transcriptional study, the p. alvei chromosome should be sequenced.
also, one of future work would be to study which stage of the sporulation cascade or what genetic mechanism is being affected by indole.
for example, it is interesting to find indole-interacting proteins in p. alvei, as previously identified indole-binding pyka of s. aurantiaca  <cit> .
endospore formation is an altruistic behavior of mother cells that provides the maximum chance of survival for the group  over any its neighbor species  <cit> .
however, the formation of an environmentally resistant spore of pathogenic bacteria, such as bacillus anthracis and various clostridium app., are problematic to human health  <cit> .
hence it is important to find a tool which controls sporulation as a disinfectant or sporocide.
the current study has revealed the natural action of sporulation reduction by indole and the plant auxin 3-indolylacetonitrile.
previously, 3-indolylacetonitrile from cruciferous vegetables , such as broccoli, cauliflower, and cabbage, was seen to decrease the biofilm formation of two pathogenic bacteria, e. coli o157:h <dig> and p. aeruginosa by inhibiting polymeric matrix production  <cit> .
hence, indole and 3-indolylacetonitrile are possible spore maturation inhibitors against spore-forming p. alvei and biofilm inhibitors against pathogenic biofilm formation.
currently, various indole derivatives from plants and numerous synthetic indole derivatives are commercially available and work is in progress to identify universal and stronger sporocides and to understand their genetic mechanism in action.
CONCLUSIONS
the current study demonstrates that i) indole is an extracellular stationary phase molecule in a gram-positive bacteria p. alvei, ii) indole clearly inhibits spore maturation and survival rates under several stresses in p. alvei without affecting cell growth, iii) plant auxin 3-indolylacetonitrile dramatically decreased the heat resistance of p. alvei, iv) electron microscopy shows that indole and 3-indolylacetonitrile inhibit the development of spore coats and cortex in p. alvei.
this study shows that indole, as a signaling molecule in quorum-sensing manner, plays a role in sporulation of p. alvei and that 3-indolylacetonitrile can be useful to control of heat and antimicrobial resistant spores of gram-positive bacteria.

 in nature, a key element of the adaptive responses of bacteria is the ability to sense and respond to the local environment, such as nutritional limitation, their population, the presence of toxic chemicals from other bacteria and host signals. hence, it is important to coordinate the pattern of gene expression, and bacteria have evolved specific mechanisms to ensure the survival of the species in environmental niches. for example, many bacteria use a variety of intercellular signaling systems including quorum sensing. the intercellular signal molecules include n-acyl-homoserine lactones in gram-negative bacteria, autoinducer and indole in both gram-negative and gram-positive bacteria, signal peptides in gram-positive bacteria, and others; these have been seen to co-ordinate gene expression for bioluminescence, sporulation, plasmid conjugal transfer, competence, virulence factor production, antibiotic production, and biofilm formation. indole is an intercellular signal as well as an interspecies signal. a variety of both gram-positive and gram-negative bacteria produce indole using tryptophanase that can reversibly convert tryptophan into indole, pyruvate, and ammonia according to reaction below. indole plays diverse biological roles in the microbial community; for example, indole controls the virulence, biofilm formation, acid resistance, and drug resistance in gram-negative bacteria. in a gram-positive stigmatella aurantiaca, indole increases its sporulation via indole binding pyruvate kinase. moreover, recent studies suggest that abundant bacterial indole in human intestines plays beneficial roles in the human immune system. also importantly, indole increases escherichia coli antibiotic resistance, which eventually leads to population-wide resistance. p. alvei belongs to the class bacillales, which includes bacillus, listeria, and staphylococcus and is an endospore-forming gram-positive bacterium that swarms on routine culture medium. p. alvei is frequently present in cases of european foulbrood and has, on occasion, been the cause of human infections. p. alvei is the only indole-producing bacterium among many bacillus species, and the biosynthesis of indole has been well-studied in p. alvei. it has long been thought that indole producing bacteria including p. alvei utilize tryptophanase to synthesize tryptophan and other amino acids from indole as a carbon source. however, the equilibrium of the reaction favors the production of indole from tryptophan. hence, we sought here the real biological role of indole in p. alvei physiology. spore-forming bacteria can respond to nutritional limitation and harsh environmental conditions by forming endospores that are remarkably resistant to heat, desiccation, and various chemicals. spore formation is an elaborate and energy intensive process that requires several hours to complete. therefore, sporulation is a last-resort adaptive process that is tightly regulated by complex cell-cell signaling or so-called quorum sensing. bacillus subtilis produces multiple cell-cell signaling molecules to control the sophisticated sporulation that is often a temporal, spatial, and dynamic decision-making process. the outermost protective layers of b. subtilis endospores are the coat and the cortex. the spore coat is a barrier against bactericidal enzymes and destructive chemicals. therefore, heat resistant spores are also resistant to treatment by various chemicals, such as acids, bases, oxidizing agents, alkylating agents, aldehydes and organic solvents. thus, we investigated the role of indole on heat resistance as well as other environmental stresses. in this study, we identified that indole was a stationary phase extracellular molecule in p. alvei and functioned to inhibit spore maturation and to decrease survival rates under several environmental stresses. additionally, we studied the effect of indole derivatives originated from plants on spore formation in p. alvei. this study provides another important role of indole and indole derivatives.  extracellular indole accumulation in p. alvei to be an environmental signal molecule, indole has to be excreted out of cells. thus, the cell growth of p. alvei and the extracellular indole concentration were measured in luria-bertani medium. clearly, the level of extracellular indole from p. alvei was growth-dependent. indole production was begun in the middle of exponential growth phase and reached the maximum amount in the stationary phase. notably, the level of extracellular indole present was stable over time at 37°c, which was one of characteristics of the indole molecule while other signaling molecules, such as ahls, ai- and signal peptides, are only temporally present and heat-unstable. the accumulation pattern of extracellular indole was similar to that of other bacteria, such as e. coli and vibrio cholera, while these two bacteria accumulated up to 500- μm of extracellular indole within h in lb. the slower accumulation of indole in p. alvei was probably due to the 200-fold lower activity of p. alvei tryptophanase than that of e. coli tryptophanase. catabolite repression of p. alvei tryptophanase since indole production was suppressed by the presence of glucose in e. coli through catabolite repression, the effect of carbon sources on indole production was investigated in p. alvei. similar to e. coli, the addition of glucose and glycerol in lb medium completely abolished the production of indole in p. alvei for h, while lactose did not affect indole accumulation. this result suggested that the indole accumulation in p. alvei was strictly controlled by catabolic repression although transport mechanisms of glucose and glycerol would be different. in other words, p. alvei did not produce indole in the presence of the preferred carbon sources such as glucose and glycerol. unlike the current observation, it was previously reported that the tryptophanase in b. alvei appeared to be constitutive, and catabolite repression was not operative. the report studied the effect of only tryptophan on tryptophanase activity and found that the activity of p. alvei tryptophanase was independent of tryptophan. indole inhibits the heat-resistant cell numbers of p. alvei the main hypothesis of this study was that a large quantity of extracellular indole would play a quorum sensing role in cell physiology of p. alvei so we investigated the effect of indole on sporulation and biofilm formation which was influenced by cell population and environmental stresses in other bacillus strains. in p. alvei, the addition of exogenous indole surprisingly decreases the heat-resistant colony-forming unit in a dose dependent manner. for example, indole decreased the heat-resistant cfu of p. alvei compared to no addition of indole 51-fold at hr and 10-fold at hr. to confirm the presence of exogenous indole, the indole level in dsm medium was measured with hplc. the level of exogenous indole was not changed at all over h. hence, the exogenous indole was not utilized as a carbon source and inhibited the heat-resistant cfu of p. alvei. additionally, the temperature effect of indole on the heat resistance of p. alvei was investigated since the environmental temperature affected indole signaling in e. coli. unlike in e. coli, the inhibitory effect of indole on the heat-resistant cfu of p. alvei at 30°c was similar to that at 37°c in p. alvei. hence, it appeared that the temperature effect of indole on the heat-resistant cfu of p. alvei was not significant under the tested laboratory conditions. indole inhibits the development of spore coat and cortex the effect of indole on the morphology of sporulating cells was examined by transmission electron microscopy. surprisingly, the proportion of sporulating cells in the total number of cells was similar between with and without treatment of indole. however, exogenous addition of indole influenced the morphology of the spore coat and the cortex. cells with exogenous indole formed endospores with a thin spore coat and a thin spore cortex, while using no indole treatment resulted in a thick spore coat and cortex. because the spore coat and cortex were important for heat resistance and chemical resistance, we concluded that indole caused an immature spore that negatively contributed to the heat resistance of p. alvei. effect of indole derivatives on the heat resistance of p. alvei in the natural environment, indole can be easily oxidized into hydroxyindoles by diverse oxygenases, and indole derivatives often show different effects on bacterial physiology. thus, p. alvei can often encounter many kinds of indole-like compounds that are synthesized from tryptophan in other bacteria, plants, and even animals. therefore, seven indole derivatives have been further investigated for the heat resistance of p. alvei. as a negative control, glucose was used since glucose decreased the sporulation of b. subtilis. similar to b. subtilis, glucose clearly decreased the heat-resistant cfu by 600-fold in p. alvei. however, l-tryptophan as the main substrate of the indole biosynthesis did not have much influence on the heat-resistant cfu, which supported that indole rather than tryptophan specifically influenced the heat resistance of p. alvei. most interestingly, a plant auxin, 3-indolylacetonitrile dramatically decreased the heat-resistant cfu of p. alvei in a dose dependent manner at and hr, while another auxin 3-indoleacetic acid had a less significant influence, and tryptamine and 2-oxindole had no effect. therefore, these results suggest that the functional groups of indole derivatives may control the development of p. alvei spores. similar to indole, the proportion of sporulating cells in the total number of cells was similar with and without treatment of 3-indolylacetonitrile. also, 3-indolylacetonitrile produced an irregular spore coat, while no treatment produced sturdy coat. therefore, it appeared that indole and 3-indolylacetonitrile inhibited spore maturation rather than sporulation initiation. in order to understand how most spores in the presence of indole and 3-indolylacetonitrile could not survive against heat treatment, the lysozyme resistance assay was performed with 30-hour grown cells since the lysozyme treatment could release all spores. as a result, indole and 3-indolylacetonitrile produced a large portion of lysozyme-resistant cells which are probably the number of total spores, while indole and 3-indolylacetonitrile produced only ± % and ± % heat-resistant cells; hence it appeared that a large number of spores have some spore defect for heat resistance. therefore, it appeared that the low heat-resistant cfu was caused by some spore defect or the altered spore structure. furthermore, the effect of indole and 3-indolylacetonitrile was investigated using another spore-forming medium, brain heart infusion agar for a longer incubation time when sporulation process would be completed. similar to dsm medium, indole and 3-indolylacetonitrile inhibited the heat-resistant cfu of p. alvei, compared to no addition of exogenous indole. therefore, the inhibitory impact of indole and 3-indolylacetonitrile was effective in different media for a long term, while their effect on heat resistance was attenuated with a longer incubation time. effect of indole and indole derivatives on cell growth to test the toxicity of indole and indole derivatives, cell turbidity at hr and the specific growth rates with indole and 3-indolylacetonitrile were measured. most indole derivatives at the concentration tested did not have much of an inhibition effect on the cell growth of p. alvei, while indole-3-acetamide and 2-oxindole slightly decreased cell growth. the growth rate of p. alvei was ± /h in the absence of the indole derivatives in lb medium, whereas the growth rate was ± /h with indole and ± /h with 3-indolylacetonitrile. in dsm medium, the growth rate of p. alvei was ± /h in the absence of the indole derivatives, whereas the growth rate was ± /h with indole and ± /h with 3-indolylacetonitrile. therefore, indole and 3-indolylacetonitrile were not toxic to p. alvei and the inhibitory effect of the heat resistance was mostly due to the function of indole and 3-indolylacetonitrile rather than growth inhibition. indole contributes to low survival against environmental stresses since endospores are remarkably resistant to heat as well as various chemicals, we presumed that indole also decreased the resistance to environmental stresses, such as treatment with antibiotics, ethanol and low ph. as expected, indole decreased the survival rates with three antibiotics and when exposed to low ph and 70% ethanol. for example, indole decreased tetracycline resistance -fold, erythromycin resistance -fold, and chloramphenicol resistance 4-fold, and the survival rates with ethanol -fold and ph 21-fold, respectively. these results are a good match with the sporulation results. effect of indole on the survival of b. subtilis spores since p. alvei belongs to the same bacillales order including b. subtilis, the effect of indole and 3-indolylacetonitrile was investigated in b. subtilis that did not produce indole. unlike p. alvei, indole and 3-indolylacetonitrile had no impact on the heat resistance in b. subtilis, while glucose treatment as a negative control significantly decreased the heat-resistant cfu. hence, it appeared that the action mechanism of indole was different between indole-producing p. alvei and non-indole-producing b. subtilis. discussion indole is an abundant environmental signal in both gram-positive and gram-negative bacteria. currently, the diverse roles of indole as an intercellular signal are beginning to be revealed in various indole-producing-bacteria, such as e. coli, vibrio cholerae, stigmatella aurantiaca, fusobacterium nuceatum, and porphyromonas gingivalis, as well as in non-indole-producing bacteria, such as pseudomonas aeruginosa and salmonella enterica. the current study shows that the environmental signal indole also has a role in gram-positive p. alvei. interestingly, the role of indole seems to be substantially divergent in different microorganisms, reflecting adaptation to different environments and niche-specific challenges. for example, indole differently controls biofilm formation in different e. coli strains, vibrio cholerae, and fusobacterium nuceatum. also, indole and indole derivatives induced sporulation in stigmatella aurantiaca, while this study shows that indole reduced the integrity of spores in p. alvei. therefore, the results suggest that different bacterial species have developed their unique systems to beneficially utilize indole in their microbial community. previously, it was reported that indole derivatives, such as 3-indoleacetic acid, 3-indolylacetonitrile, tryptamine, and 2-oxindole, but not indole, decreased the percentages of spore germination and appressorium formation, which inhibited all stages of infection behaviors in a rice pathogen magnaporthe grisea. these results and the current study suggest that indole derivatives, such as 3-indolylacetonitrile, can be used as protective compounds against spore-forming p. alvei. since indole influenced the biofilm formation of several indole-producing bacteria, such as e. coli, vibrio cholerae, and fusobacterium nuceatum, and the sporulation transcription factor spoa was required for biofilm development in b. subtilis, the effect of indole on the biofilm formation of p. alvei was investigated. however, indole did not show an effect on p. alvei biofilm formation in the 96-well plate biofilm assay in lb or dsm media either at 30°c and at 37°c. therefore, the indole-involving mechanism of p. alvei biofilm formation is different from that in other strains. glucose obviously prevented the development of cfu of p. alvei presumably by preventing sporulation as well as in b. subtilis via catabolite repression. because indole is a stationary phase signal and because the production of indole is tightly regulated by carbon sources, the role of indole on spore formation could be closely affected by the catabolite repression. thus, indole serves not only as an indicator of cell population, but also as an indicator of starvation. this dual function of indole may reflect the status of cells in the environment. because the accumulation of extracellular indole can be dramatically affected by many environmental factors in addition to carbon sources, the action of indole would be governed by the environment in a sophisticated manner. nevertheless, the question remains as to why p. alvei produces copious amount of extracellular indole, as it causes immature spore formation. one possible explanation can be found in the previous study in that bacteria utilize indole as a defense tool against non-indole producing pathogenic p. aeruginosa to diminish its virulence. another possible answer is that indole intentionally lowers integrity of spores in order to make cells easy to resume growth when the environment is favorable again at a later date. hence, a large quantity of indole is an indicator of a favorable environment in which other unfavorable species are scare and indole may control the timing of germination in natural environments. although highly speculative, another possibility is that indole signal negatively controls spore maturation, while other quorum sensing molecules positively regulates sporulation of bacillus, even using multiple signaling molecules. also, there is the possibility that indole is affecting spore germination since indole lowered the survival against environmental stresses while the number of spore was not affected by indole. however, it is unclear, so far, how the indole signal influences sporulation in p. alvei. it is necessary to identify the operon of p. alvei tryptophanase to understand the genetic regulation of indole biosynthesis. for further transcriptional study, the p. alvei chromosome should be sequenced. also, one of future work would be to study which stage of the sporulation cascade or what genetic mechanism is being affected by indole. for example, it is interesting to find indole-interacting proteins in p. alvei, as previously identified indole-binding pyka of s. aurantiaca. endospore formation is an altruistic behavior of mother cells that provides the maximum chance of survival for the group over any its neighbor species. however, the formation of an environmentally resistant spore of pathogenic bacteria, such as bacillus anthracis and various clostridium app., are problematic to human health. hence it is important to find a tool which controls sporulation as a disinfectant or sporocide. the current study has revealed the natural action of sporulation reduction by indole and the plant auxin 3-indolylacetonitrile. previously, 3-indolylacetonitrile from cruciferous vegetables, such as broccoli, cauliflower, and cabbage, was seen to decrease the biofilm formation of two pathogenic bacteria, e. coli o157:h and p. aeruginosa by inhibiting polymeric matrix production. hence, indole and 3-indolylacetonitrile are possible spore maturation inhibitors against spore-forming p. alvei and biofilm inhibitors against pathogenic biofilm formation. currently, various indole derivatives from plants and numerous synthetic indole derivatives are commercially available and work is in progress to identify universal and stronger sporocides and to understand their genetic mechanism in action.  the current study demonstrates that i) indole is an extracellular stationary phase molecule in a gram-positive bacteria p. alvei, ii) indole clearly inhibits spore maturation and survival rates under several stresses in p. alvei without affecting cell growth, iii) plant auxin 3-indolylacetonitrile dramatically decreased the heat resistance of p. alvei, iv) electron microscopy shows that indole and 3-indolylacetonitrile inhibit the development of spore coats and cortex in p. alvei. this study shows that indole, as a signaling molecule in quorum-sensing manner, plays a role in sporulation of p. alvei and that 3-indolylacetonitrile can be useful to control of heat and antimicrobial resistant spores of gram-positive bacteria. 
unlike previous studies, our data show that the production of indole in p. alvei is strictly controlled by catabolite repression since the addition of glucose and glycerol completely turns off the indole production. in this study, we sought a novel role of indole in a gram-positive bacteria paenibacillus alvei that can produce extracellular indole at a concentration of up to μm in the stationary phase in luria-bertani medium. together, our results demonstrate that indole and plant auxin 3-indolylacetonitrile inhibit spore maturation of p. alvei and that 3-indolylacetonitrile presents an opportunity for the control of heat and antimicrobial resistant spores of gram-positive bacteria. the addition of exogenous indole markedly inhibits the heat resistance of p. alvei without affecting cell growth. observation of cell morphology with electron microscopy shows that indole inhibits the development of spore coats and cortex in p. alvei. a variety of both gram-positive and gram-negative bacteria produce large quantities of indole that functions as an intercellular signal controlling diverse aspects of bacterial physiology. additionally, indole derivatives also influence the heat resistance; for example, a plant auxin, 3-indolylacetonitrile dramatically decreased the heat resistance of p. alvei, while another auxin 3-indoleacetic acid had a less significant influence on the heat resistance of p. alvei. as a result of the immature spore formation of p. alvei, indole also decreases p. alvei survival when exposed to antibiotics, low ph, and ethanol. bacteria use diverse signaling molecules to ensure the survival of the species in environmental niches.
 bacteria use diverse signaling molecules to ensure the survival of the species in environmental niches. a variety of both gram-positive and gram-negative bacteria produce large quantities of indole that functions as an intercellular signal controlling diverse aspects of bacterial physiology.  in this study, we sought a novel role of indole in a gram-positive bacteria paenibacillus alvei that can produce extracellular indole at a concentration of up to μm in the stationary phase in luria-bertani medium. unlike previous studies, our data show that the production of indole in p. alvei is strictly controlled by catabolite repression since the addition of glucose and glycerol completely turns off the indole production. the addition of exogenous indole markedly inhibits the heat resistance of p. alvei without affecting cell growth. observation of cell morphology with electron microscopy shows that indole inhibits the development of spore coats and cortex in p. alvei. as a result of the immature spore formation of p. alvei, indole also decreases p. alvei survival when exposed to antibiotics, low ph, and ethanol. additionally, indole derivatives also influence the heat resistance; for example, a plant auxin, 3-indolylacetonitrile dramatically decreased the heat resistance of p. alvei, while another auxin 3-indoleacetic acid had a less significant influence on the heat resistance of p. alvei.  together, our results demonstrate that indole and plant auxin 3-indolylacetonitrile inhibit spore maturation of p. alvei and that 3-indolylacetonitrile presents an opportunity for the control of heat and antimicrobial resistant spores of gram-positive bacteria. 