BACKGROUND
spores of bacillus licheniformis and other bacillus species are frequent contaminants in foods
<cit> .
exposure to nutrients triggers spores to leave dormancy in the process of germination
<cit> .
this process involves several steps leading to rehydration of the spore core and loss of dormancy.
under favorable conditions, spores will grow out and multiply to numbers that can cause food spoilage and sometimes disease
<cit> .
while dormant spores are extremely heat resistant, germinated spores can easily be killed by a mild heat treatment
<cit> .
therefore, a food preservation technique applied by food manufacturers to reduce spore numbers in food products is “induced germination”.
the consequence of induced germination is spores germinated into vegetative cells will be heat sensitive and can therefore be inactivated, by successive heating below temperatures that compromise food quality
<cit> .
the effectiveness of such a strategy depends on the germination rate of the spore population.
a slow and/or heterogeneous germination rate of a specific spore population will reduce the effectiveness of such treatments
<cit> .
nutrient germinant receptors , localized to the inner spore membrane
<cit> , are involved in the spore’s recognition of specific nutrients in its environment, which is the initial step in the spore’s return to growth
<cit> .
binding of nutrient to the receptors is believed to trigger the release of the spore core’s large depot of ca-dipicolinic acid , followed by rehydration of the spore core and degradation of the spore cortex
<cit> .
current knowledge about grs and their nutrient specificity is mainly achieved from bacillus subtilis and bacillus cereus.
however, genes encoding grs are widely distributed among bacillus and clostridium species
<cit> , implicating an essential role in triggering of spore germination in most spore-forming bacteria.
interestingly, the nutrient specificity of the receptors and the interaction between them varies between and even within species, as has been shown for b. cereus-group members
<cit> .
grs are generally encoded by polycistronic operons that are expressed late in sporulation under the regulation of the forespore-specific transcription factor, sigma g
<cit> .
these genes constitute a family  of homologous genes that probably have evolved from the same ancestor
<cit> .
three putative gera family operons, gera , gerk  and ynd  and the single gerac homologue yndf <dig> have been identified within the b. licheniformis type strain atcc14580/dsm <dig> genome
<cit> .
of these, only the gera operon has been functionally characterized so far
<cit> .
gera was found to be essential for germination in presence of l-alanine.
a similar role has been described for gera in b. subtilis <cit> .
l-alanine is probably the most universal single nutrient germinant among spore formers
<cit> .
the bacillus grs which have been described so far are usually composed of three subunits termed a, b and c. the a and b subunits are predicted to contain 5– <dig>  and 10– <dig>  membrane-spanning domains, respectively
<cit> , while the c subunit is thought to be a membrane-anchored lipoprotein
<cit> .
the tertiary structure of b. subtilis gerbc was determined a few years ago
<cit> .
the b-subunit, whose amino acid sequence shows homology to proteins of the apc  superfamily, is proposed to be the most likely site of ligand binding, as mutations within this subunit alter ligand specificity
<cit> .
however, since mutations in any of the three cistrons are shown to disturb receptor function, the exact site of nutrient binding is still unknown
<cit> .
the genetic relationship of  <dig> strains of the food-spoilage agent b. licheniformis, a close relative of b. subtilis, was recently described by a novel mlst scheme
<cit> .
one of these strains, nvh <dig>  was isolated after surviving an “induced germination”-regime , applied by the food industry to eliminate spore contamination.
preliminary results in our lab suggested that nvh <dig> and other b. licheniformis strains germinate considerably slower than the type strain when exposed to l-alanine.
such slow-germinating strains pose a challenge to food manufacturers that want to implement “induced germination” as a strategy to reduce/eliminate spores during processing.
in this study,  <dig> of the  <dig> genotyped strains were screened for efficiency of l-alanine-induced germination, and the correlation between the genotype and the induced germination was determined.
furthermore, it was investigated whether the slow germination of three particular b. licheniformis strains was due to sequence differences in the gera operon.
RESULTS
screening of l-alanine-induced germination in b. licheniformis strains
in order to evaluate the efficiency of l-alanine-induced germination of the  <dig> b. licheniformis strains, the level of germination was recorded after addition of l-alanine in a screening assay.
the results showed that germination efficiency, determined by reduction of absorbance  varied from ~ <dig> to 60% between the tested strains 2 h after the addition of germinant .
a drop in a <dig> of 60% was equivalent to > 95% germinated spores, as verified by phase contrast microscopy.
about  <dig> of the strains germinated well with a reduction in absorbance of 40% or more, while six strains germinated poorly .
in general, differences in germination between strains may be due to differences in lag time  and differences in rate of germination .
several factors may account for these differences:  permeability of the outer spore layers, restricting access of germinant to the inner membrane
<cit> ,  germinant specificity
<cit> ,  gr  level
<cit> ,  dysfunctional grs
<cit> ,  gr synergism/antagonism
<cit>  and/or  structure of the cortex
<cit> .
within single populations of b. subtilis, a reduced level of grs has been suggested to be one of the main reasons for slow germination or “superdormancy”
<cit> , probably by increasing the lag time until cadpa is released
<cit> .
in b. subtilis, grs have been proposed to be present in a relatively low number  in the spore’s inner membrane where they form discrete clusters, so-called germinosomes
<cit> , however, it has recently been reported that this number may be highly underestimated
<cit> .
the number of germination receptors has been shown to be strongly dependent on the sporulation conditions
<cit> .
in this study, sporulation and germination conditions  were optimized with respect to the type strain atcc14580/dsm <dig>  however, these conditions may not be optimal for all strains.
distribution and characterization of the gera operon
the gera locus was detected by pcr in all of the  <dig> genotyped b. licheniformis strains .
to investigate whether certain gera sequence variants were associated with slow germination, partial gera operon sequences of all strains were analysed, aligned and organized into clusters.
the resulting neighbour-joining  tree is presented in figure
<dig>  with the exception of two strains  the nj- dendogram was congruent with the mlst tree generated from six house-keeping genes
<cit> .
thus, the gera locus seemed to have evolved in parallel to the house-keeping genes.
the ratio of non-synonymous versus synonymous base substitutions  was  <dig>  which is somewhat higher than the calculated values for the individual mlst loci
<cit> , but far below the limit of  <dig>  that is often set for loci undergoing positive selection.
thus, the gera locus, similar to the house-keeping genes, seems to be subject to purifying  selection
<cit> .
a total of seven unique alleles were distributed into four main clusters, determined “1a”, “1b”, “1c” and “2” .
cluster “2” was represented by only three strains, nvh <dig>  nvh <dig> and nvh <dig>  that all showed a slower and less efficient germination response  compared to the type strain, atcc14580/dsm <dig> .
however, slow-germinating strains were also found within each of the other clusters.
thus, this part of the gera operon sequence  was not suitable in order to completely distinguish slow-germinating and fast-germinating strains.
germination of gera complementation strains
in order to further investigate the influence of gera sequences on germination rate, mw3Δgeraa was complemented with gera operons originating from the type strain atcc14580/dsm13
<cit> , and the three slow-germinating strains .
the gera sequences of atcc14580/dsm <dig> , nvh <dig> and nvh <dig> nearly restored the phenotype of the sequence originating strains, while complementing mw3∆geraa with the gera sequence from nvh <dig> increased the germination rate of the complemented strain compared to nvh <dig> wild-type .
still, the order of the germination rate between the four strains was consistent between the two experiments , substantiating that the phenotypes of the complemented mw3∆geraa mutant to some extent restored the phenotypes of the gera originating strains.
germination data of mw <dig> carrying pht <dig>  showed that carrying the empty vector, or the use of erythromycin in the cultures, hampered the germination rate of the mw <dig> strain .
however, we assume that comparing the effect of the complementing sequences is acceptable since they are all carried by the same vector.
3d for comparison.
an important observation was that, in contrast to løvdal et al.
2012
<cit> , l-alanine-induced germination was not completely abolished in mw3∆geraa .
this weak germination  was not observed in absence of germinant, indicating that germination receptors other than gera might be weakly activated by l-alanine.
we also noted that spores of the slow-germinating strain nvh <dig> hardly germinated at all, and to a lesser extent than mw3∆geraa .
when complementing mw3∆geraa with the gera operon from nvh <dig>  germination efficiency increased, indicating that the gera operon of nvh <dig> has some functionality in presence of l-alanine.
a faster and more efficient germination of the complementation mutants compared to their respectively gera originating strains was also observed for nvh <dig>  and nvh  <dig> .
the imperfect complementation of the phenotypes may be due to several different factors.
firstly, a two- to seventeen-fold increase in expression level of geraa was observed when mw3∆geraa was complemented with different gera sequences and compared to the wild-type strains from where the gera sequences originated .
the increased geraa expression level in the complementation mutants might be related to the copy-number of the plasmid pht <dig> .
previous experiments have shown that a 2– <dig> fold overexpression of ger genes may increase germination rate
<cit> .
secondly, since the complementing gera genes in this experiment were plasmid-born , 1 μg ml- <dig> erythromycin was used in the sporulation medium to maintain the plasmid throughout the sporulation process and mw <dig> carrying the pht <dig> empty vector germinated slower and with less efficiency than the wild-type mw <dig> strain .
despite this observation, mw3∆geraa complemented with slow germinating gera sequences germinated better than the strains from where the gera sequences originated .
thirdly, the entire gera operon and the 151 bp region upstream of the start codon of geraa was cloned in the complementing vector pht <dig>  alignments of the promoter sequence of strain nvh <dig>  nvh <dig>  nvh <dig> and atcc14580/mw <dig> can be viewed in additional file
<dig>  no differences were observed between the type-strain and the slow germinating strains in the - <dig> and - <dig> promoter region of gera.
however, differences outside these regions may influence the transcriptional level.
pht315
<cit>  contains the inducible lac promoter, but transcription from this promoter cannot be excluded even without induction.
despite the imperfect restoration of the wt phenotypes, the results of the germination assays in this study indicate that the gera sequences have an impact on germination rate and efficiency.
differences in the gera amino acid sequence may lead to altered protein 3-d structure, which again may cause impaired assembly and stability of the receptor complex in the inner membrane, lower or higher substrate affinity or influence the interactions with other membrane proteins.
structural analysis of amino acid substitutions in the gera receptor
analyses of single amino acid substitutions have previously been conducted in b. subtilis geraa
<cit> , gerab
<cit>  and gerbc
<cit> .
none of these positions were substituted in the gera sequences examined in the present study.
alignments of the geraa, gerab and gerac sequences of b. licheniformis nvh <dig>  nvh <dig>  nvh <dig> and atcc14580/dsm <dig> are presented in additional files
<dig>
<dig> and
<dig>  thus, on the basis of this knowledge and the lack of a 3d structure of any proteins in the geraa and gerab families of proteins, the relevance of the observed differences within these two subunits is difficult to determine.
however, the crystal structure of b. subtilis gerbc has recently been determined
<cit> .
using this structure as a template for prediction of b. licheniformis gerac structures, one of the perhaps most interesting substitutions is f342s  which lies in the so-called “region 2” of domain iii
<cit>  .
region  <dig> is reported to be a well conserved region in gerbc among bacillales and substitutions within this region were previously shown to affect receptor function in b. subtilis <cit> .
on the other hand, the f342s substitution was neither observed in the gera sequences of the slowest germinating strain nvh <dig> or the fastest germinating strain atcc14580/dsm <dig> suggesting that the role of this site seems unclear.
it should be mentioned that the aa sequence of the gerac protein of nvh <dig> is much closer to that of mw <dig> than the other two , indicating that gerac is not crucial to germination efficiency.
ultimately, the lack of information about the exact germinant binding site, as well as the fact that only the c subunit has been structurally characterized, makes it difficult to interpret the effect of single substitutions on the gera receptor function.
CONCLUSIONS
this study shows that spores of  <dig> b. licheniformis strains are able to germinate in the presence of l-alanine, but that the germination rate and efficiency differ significantly between the strains.
about 10% of the strains germinated poorly, even in presence of high  concentrations of probably the most universal and potent germinant for bacillus species in general, and b. licheniformis in particular.
germination rate of different bacterial strains are of importance to the food industry, using so-called “induced germination”, eg tyndallization, to decrease spore contamination in processed foods.
delayed germination may reduce the efficiency of tyndallization by allowing ungerminated spores to survive.
our results demonstrate that nutrient-induced germination followed by inactivation can be challenging when dealing with specific b. licheniformis strains.
the germination phenotype was partly restored when complementing a geraa disruption mutant with gera operons from either slow- or fast-germinating b. licheniformis strains.
this observation indicates that differences in gera family operons are partly responsible for differences in germination efficiency of b. licheniformis in response to l-alanine.

 spores of bacillus licheniformis and other bacillus species are frequent contaminants in foods. exposure to nutrients triggers spores to leave dormancy in the process of germination. this process involves several steps leading to rehydration of the spore core and loss of dormancy. under favorable conditions, spores will grow out and multiply to numbers that can cause food spoilage and sometimes disease. while dormant spores are extremely heat resistant, germinated spores can easily be killed by a mild heat treatment. therefore, a food preservation technique applied by food manufacturers to reduce spore numbers in food products is “induced germination”. the consequence of induced germination is spores germinated into vegetative cells will be heat sensitive and can therefore be inactivated, by successive heating below temperatures that compromise food quality. the effectiveness of such a strategy depends on the germination rate of the spore population. a slow and/or heterogeneous germination rate of a specific spore population will reduce the effectiveness of such treatments. nutrient germinant receptors, localized to the inner spore membrane, are involved in the spore’s recognition of specific nutrients in its environment, which is the initial step in the spore’s return to growth. binding of nutrient to the receptors is believed to trigger the release of the spore core’s large depot of ca-dipicolinic acid, followed by rehydration of the spore core and degradation of the spore cortex. current knowledge about grs and their nutrient specificity is mainly achieved from bacillus subtilis and bacillus cereus. however, genes encoding grs are widely distributed among bacillus and clostridium species, implicating an essential role in triggering of spore germination in most spore-forming bacteria. interestingly, the nutrient specificity of the receptors and the interaction between them varies between and even within species, as has been shown for b. cereus-group members. grs are generally encoded by polycistronic operons that are expressed late in sporulation under the regulation of the forespore-specific transcription factor, sigma g. these genes constitute a family of homologous genes that probably have evolved from the same ancestor. three putative gera family operons, gera, gerk and ynd and the single gerac homologue yndf have been identified within the b. licheniformis type strain atcc14580/dsm genome. of these, only the gera operon has been functionally characterized so far. gera was found to be essential for germination in presence of l-alanine. a similar role has been described for gera in b. subtilis. l-alanine is probably the most universal single nutrient germinant among spore formers. the bacillus grs which have been described so far are usually composed of three subunits termed a, b and c. the a and b subunits are predicted to contain 5– and 10– membrane-spanning domains, respectively, while the c subunit is thought to be a membrane-anchored lipoprotein. the tertiary structure of b. subtilis gerbc was determined a few years ago. the b-subunit, whose amino acid sequence shows homology to proteins of the apc superfamily, is proposed to be the most likely site of ligand binding, as mutations within this subunit alter ligand specificity. however, since mutations in any of the three cistrons are shown to disturb receptor function, the exact site of nutrient binding is still unknown. the genetic relationship of strains of the food-spoilage agent b. licheniformis, a close relative of b. subtilis, was recently described by a novel mlst scheme. one of these strains, nvh was isolated after surviving an “induced germination”-regime, applied by the food industry to eliminate spore contamination. preliminary results in our lab suggested that nvh and other b. licheniformis strains germinate considerably slower than the type strain when exposed to l-alanine. such slow-germinating strains pose a challenge to food manufacturers that want to implement “induced germination” as a strategy to reduce/eliminate spores during processing. in this study, of the genotyped strains were screened for efficiency of l-alanine-induced germination, and the correlation between the genotype and the induced germination was determined. furthermore, it was investigated whether the slow germination of three particular b. licheniformis strains was due to sequence differences in the gera operon.  screening of l-alanine-induced germination in b. licheniformis strains in order to evaluate the efficiency of l-alanine-induced germination of the b. licheniformis strains, the level of germination was recorded after addition of l-alanine in a screening assay. the results showed that germination efficiency, determined by reduction of absorbance varied from ~ to 60% between the tested strains 2 h after the addition of germinant. a drop in a of 60% was equivalent to > 95% germinated spores, as verified by phase contrast microscopy. about of the strains germinated well with a reduction in absorbance of 40% or more, while six strains germinated poorly. in general, differences in germination between strains may be due to differences in lag time and differences in rate of germination. several factors may account for these differences: permeability of the outer spore layers, restricting access of germinant to the inner membrane, germinant specificity, gr level, dysfunctional grs, gr synergism/antagonism and/or structure of the cortex. within single populations of b. subtilis, a reduced level of grs has been suggested to be one of the main reasons for slow germination or “superdormancy”, probably by increasing the lag time until cadpa is released. in b. subtilis, grs have been proposed to be present in a relatively low number in the spore’s inner membrane where they form discrete clusters, so-called germinosomes, however, it has recently been reported that this number may be highly underestimated. the number of germination receptors has been shown to be strongly dependent on the sporulation conditions. in this study, sporulation and germination conditions were optimized with respect to the type strain atcc14580/dsm however, these conditions may not be optimal for all strains. distribution and characterization of the gera operon the gera locus was detected by pcr in all of the genotyped b. licheniformis strains. to investigate whether certain gera sequence variants were associated with slow germination, partial gera operon sequences of all strains were analysed, aligned and organized into clusters. the resulting neighbour-joining tree is presented in figure  with the exception of two strains the nj- dendogram was congruent with the mlst tree generated from six house-keeping genes. thus, the gera locus seemed to have evolved in parallel to the house-keeping genes. the ratio of non-synonymous versus synonymous base substitutions was which is somewhat higher than the calculated values for the individual mlst loci, but far below the limit of that is often set for loci undergoing positive selection. thus, the gera locus, similar to the house-keeping genes, seems to be subject to purifying selection. a total of seven unique alleles were distributed into four main clusters, determined “1a”, “1b”, “1c” and “2”. cluster “2” was represented by only three strains, nvh nvh and nvh that all showed a slower and less efficient germination response compared to the type strain, atcc14580/dsm. however, slow-germinating strains were also found within each of the other clusters. thus, this part of the gera operon sequence was not suitable in order to completely distinguish slow-germinating and fast-germinating strains. germination of gera complementation strains in order to further investigate the influence of gera sequences on germination rate, mw3Δgeraa was complemented with gera operons originating from the type strain atcc14580/dsm13, and the three slow-germinating strains. the gera sequences of atcc14580/dsm, nvh and nvh nearly restored the phenotype of the sequence originating strains, while complementing mw3∆geraa with the gera sequence from nvh increased the germination rate of the complemented strain compared to nvh wild-type. still, the order of the germination rate between the four strains was consistent between the two experiments, substantiating that the phenotypes of the complemented mw3∆geraa mutant to some extent restored the phenotypes of the gera originating strains. germination data of mw carrying pht showed that carrying the empty vector, or the use of erythromycin in the cultures, hampered the germination rate of the mw strain. however, we assume that comparing the effect of the complementing sequences is acceptable since they are all carried by the same vector. 3d for comparison. an important observation was that, in contrast to løvdal et al. 2012, l-alanine-induced germination was not completely abolished in mw3∆geraa. this weak germination was not observed in absence of germinant, indicating that germination receptors other than gera might be weakly activated by l-alanine. we also noted that spores of the slow-germinating strain nvh hardly germinated at all, and to a lesser extent than mw3∆geraa. when complementing mw3∆geraa with the gera operon from nvh germination efficiency increased, indicating that the gera operon of nvh has some functionality in presence of l-alanine. a faster and more efficient germination of the complementation mutants compared to their respectively gera originating strains was also observed for nvh and nvh. the imperfect complementation of the phenotypes may be due to several different factors. firstly, a two- to seventeen-fold increase in expression level of geraa was observed when mw3∆geraa was complemented with different gera sequences and compared to the wild-type strains from where the gera sequences originated. the increased geraa expression level in the complementation mutants might be related to the copy-number of the plasmid pht. previous experiments have shown that a 2– fold overexpression of ger genes may increase germination rate. secondly, since the complementing gera genes in this experiment were plasmid-born, 1 μg ml- erythromycin was used in the sporulation medium to maintain the plasmid throughout the sporulation process and mw carrying the pht empty vector germinated slower and with less efficiency than the wild-type mw strain. despite this observation, mw3∆geraa complemented with slow germinating gera sequences germinated better than the strains from where the gera sequences originated. thirdly, the entire gera operon and the 151 bp region upstream of the start codon of geraa was cloned in the complementing vector pht alignments of the promoter sequence of strain nvh nvh nvh and atcc14580/mw can be viewed in additional file no differences were observed between the type-strain and the slow germinating strains in the - and - promoter region of gera. however, differences outside these regions may influence the transcriptional level. pht315 contains the inducible lac promoter, but transcription from this promoter cannot be excluded even without induction. despite the imperfect restoration of the wt phenotypes, the results of the germination assays in this study indicate that the gera sequences have an impact on germination rate and efficiency. differences in the gera amino acid sequence may lead to altered protein 3-d structure, which again may cause impaired assembly and stability of the receptor complex in the inner membrane, lower or higher substrate affinity or influence the interactions with other membrane proteins. structural analysis of amino acid substitutions in the gera receptor analyses of single amino acid substitutions have previously been conducted in b. subtilis geraa, gerab and gerbc. none of these positions were substituted in the gera sequences examined in the present study. alignments of the geraa, gerab and gerac sequences of b. licheniformis nvh nvh nvh and atcc14580/dsm are presented in additional files and thus, on the basis of this knowledge and the lack of a 3d structure of any proteins in the geraa and gerab families of proteins, the relevance of the observed differences within these two subunits is difficult to determine. however, the crystal structure of b. subtilis gerbc has recently been determined. using this structure as a template for prediction of b. licheniformis gerac structures, one of the perhaps most interesting substitutions is f342s which lies in the so-called “region 2” of domain iii. region is reported to be a well conserved region in gerbc among bacillales and substitutions within this region were previously shown to affect receptor function in b. subtilis. on the other hand, the f342s substitution was neither observed in the gera sequences of the slowest germinating strain nvh or the fastest germinating strain atcc14580/dsm suggesting that the role of this site seems unclear. it should be mentioned that the aa sequence of the gerac protein of nvh is much closer to that of mw than the other two, indicating that gerac is not crucial to germination efficiency. ultimately, the lack of information about the exact germinant binding site, as well as the fact that only the c subunit has been structurally characterized, makes it difficult to interpret the effect of single substitutions on the gera receptor function.  this study shows that spores of b. licheniformis strains are able to germinate in the presence of l-alanine, but that the germination rate and efficiency differ significantly between the strains. about 10% of the strains germinated poorly, even in presence of high concentrations of probably the most universal and potent germinant for bacillus species in general, and b. licheniformis in particular. germination rate of different bacterial strains are of importance to the food industry, using so-called “induced germination”, eg tyndallization, to decrease spore contamination in processed foods. delayed germination may reduce the efficiency of tyndallization by allowing ungerminated spores to survive. our results demonstrate that nutrient-induced germination followed by inactivation can be challenging when dealing with specific b. licheniformis strains. the germination phenotype was partly restored when complementing a geraa disruption mutant with gera operons from either slow- or fast-germinating b. licheniformis strains. this observation indicates that differences in gera family operons are partly responsible for differences in germination efficiency of b. licheniformis in response to l-alanine. 
in this study, we show that several of examined b. licheniformis strains germinate remarkably slower than the type strain when exposed to l-alanine. a wide selection of b. licheniformis strains was evaluated for l-alanine-induced germination efficiency. our results show that gera substitutions could only partially explain why spores of some b. licheniformis strains responded slower than others in the presence of l-alanine. three of the slow-germinating strains were further examined in order to see whether nucleotide substitutions in the gera sequences were responsible for the slow l-alanine germination. this was performed by complementing the transformable type strain derivate mw3Δgeraa with gera variants from the three slow-germinating strains; nvh nvh and nvh 
 l-alanine, acting through the gera receptor, was recently found to be an efficient germinant in bacillus licheniformis atcc14580/dsm  in this study, we show that several of examined b. licheniformis strains germinate remarkably slower than the type strain when exposed to l-alanine. these strains are not necessarily closely related, as determined by mlst. three of the slow-germinating strains were further examined in order to see whether nucleotide substitutions in the gera sequences were responsible for the slow l-alanine germination. this was performed by complementing the transformable type strain derivate mw3Δgeraa with gera variants from the three slow-germinating strains; nvh nvh and nvh  a wide selection of b. licheniformis strains was evaluated for l-alanine-induced germination efficiency. our results show that gera substitutions could only partially explain why spores of some b. licheniformis strains responded slower than others in the presence of l-alanine. bacillus licheniformisgerminationl-alaninegeragenotypegerminant receptor 