BACKGROUND
bacillus cereus is a gram positive rod-shaped aerobic, endospore-forming bacterium.
strains of b. cereus are widely distributed in the environment, mainly in soil, from where they easily spread to many types of foods, especially of vegetable origin, as well as meat, eggs, milk, and dairy products.
this bacterium is one of the leading causes of food poisoning in the developed world.
b. cereus causes two types of food-borne intoxications.
one type is characterized by nausea and vomiting and abdominal cramps and has an incubation period of  <dig> to  <dig> hours.
this is the "short-incubation" or emetic form of the disease.
the second type is manifested primarily by abdominal cramps and diarrhea with an incubation period of  <dig> to  <dig> hours.
this type is referred to as the "long-incubation" or diarrheal form of the disease  <cit> .
different strategies may be employed to prevent b. cereus poisoning, like heating food above 75°c before use to kill vegetative cells.
however, increasing trends for use of packed foods require new food preservation methods to increase the safety levels against b. cereus.
one of the current approaches is the use of antimicrobial peptides  such as enterocin as- <dig> and other bacteriocins  <cit> .
bacteriocins are small, ribosomally-synthesized antimicrobial peptides synthesized and used by one bacterium as to inhibit growth of similar or closely related bacterial strains  <cit> .
bacteriocins are categorized in several ways, e.g.
on basis of the producing strain, common resistance mechanisms, and mechanism of killing.
enterocin as- <dig> is a broad-spectrum antimicrobial peptide produced by enterococcus faecalis s- <dig>  belonging to class iii of enterococcal bacteriocins or enterocins  <cit> .
enterocin as- <dig> is a 70-residue cyclic peptide with a molecular weight of  <dig>  kda  <cit> .
the crystal structure of enterocin as- <dig> has been resolved to  <dig>  Ǻ resolution  <cit> .
it is unique with respect to its natural cyclic structure in which n and c termini are linked by a peptide bond.
it has been shown that enterocin as- <dig> adopts different oligomeric structures according to physiochemical conditions: it exists in monomeric form at ph below  <dig> and in dimeric form in the ph range of  <dig>  to  <dig> .
the molecules of as- <dig> in the crystal are arranged in chains of pairs of molecules linked either by hydrophobic interactions , or by hydrophilic interactions .
the molecules within the df-i interact through the hydrophobic helices h <dig> and h <dig>  on the other hand, the hydrophilic surfaces of helices h <dig> and h <dig> are interacting in df-ii.
the mode of action of enterocin as- <dig> has been elucidated  <cit> .
this bacteriocin makes pores of an approximate size of  <dig>  nm in the bacterial cytoplasmic membrane thereby disrupting the proton motive force and causing cell death  <cit> .
it also shows a secondary, bacteriolytic effect against some of the target bacteria.
based on its crystal structure, the proposed mechanism of action suggests that the two different stages of molecular association, df-i and df-ii, are involved in changing from the water-soluble df-i to the membrane-bound df-ii stage at the membrane surface.
this transition implies a 90° rotation of each protomer within df-i, in a way that the partially hidden hydrophobic helices h <dig> and h <dig> become solvent accessible  <cit> .
this would permit as- <dig> to insert into the bacterial membrane.
although the mechanism of action of enterocin as- <dig> has been studied extensively at physiological and physico-chemical levels, nothing is known about the responses of sensitive bacterial cells upon exposure to the bacteriocin.
previous experiment in our laboratory with as- <dig> against listeria monocytogenes showed that bacterial cells can be adapted to as- <dig>  thereby increasing resistance against as- <dig>  <cit> .
this adaptation can be achieved with subsequent inoculation in the presence of low, but still inhibitory concentrations of as- <dig>  however, the adaptation is gradually lost upon repeated subcultivation.
given the great interest of enterocin as- <dig> as a food preservative, it is of high relevance to know how the target bacteria react to bacteriocin treatment.
this may have direct implications on the elucidation of probable mechanisms for cell adaptation as well as the development of bacteriocin resistance mechanisms.
moreover, a better knowledge of the bacterial response to enterocin as- <dig> may also allow identification of new targets that could be exploited to enhance bacteriocin activity.
the purpose of the present study was to determine the genome-wide response of b. cereus cells exposed to enterocin as- <dig> and to identify components that help the bacterium to survive bacteriocin treatments.
RESULTS
effect of enterocin as- <dig> on global gene expression in b. cereus atcc14579
enterocin as- <dig> was shown to inhibit growth of vegetative cells and spore outgrowth of b. cereus  <cit>  and it can be an effective bioagent against b. cereus in various food related media, e.g.
hard cheese, rice based foods, fruit and vegetable juices  <cit> .
although the mode of action of as- <dig> is well understood, the response of bacteria to enterocin as- <dig> is poorly examined.
we have therefore determined the transcriptome of b. cereus atcc <dig> in response to as- <dig>  to omit the effect of growth inhibition related differences between the treated and the control culture, a subinhibitory bacteriocin concentration of  <dig>  μg/ml of as- <dig> was used in our experiments.
we observed no adaptation process, when b. cereus was subsequently cultivated in the presence of  <dig>  μg/ml of as- <dig>  but only when cells were treated with low, but inhibitory concentration of as- <dig> .
in preliminary experiments samples were taken  <dig> and  <dig> min after addition of as- <dig>  but we could not detect any significant changes after  <dig> min , therefore final transcriptome comparisons were performed on samples incubated with as- <dig> for  <dig> min.
transcriptome analyses were performed on six independent biological replicates.
<dig> genes were identified to be significantly upregulated, and only  <dig> genes to be downregulated .
all  <dig> downregulated genes  are located in one putative operon, coding for proteins involved in arginine and proline metabolism.
most of the upregulated genes code for proteins located in the membrane  or contain a signal sequence or periplasmic domain.
the putative membrane proteins show similarity to different transport or permease proteins or have been annotated as a hypothetical protein with no known function.
two regulators belonging to the padr family were significantly upregulated, both located upstream of, and in one operon with, genes coding for membrane proteins that are similarly enhanced during our experiments.
here, we selected the most upregulated putative operon  for further characterization.
the bc4206- <dig> operon is conserved in all fully sequenced b. cereus, b thuringiensis and b. weihenstephanensis genomes except in the b. cereus cytotoxis strain, but is missing in b. anthracis and other bacillus species.
when this operon is found in a genome, the genes surrounding this operon are also conserved .
a the ratio of gene expression is shown.
ratio: expression in as- <dig> treated sample over that in untreated samples.
b bayesian p value
c putative function of protein as annotated in the b. cereus atcc <dig> genome sequence
d domains detected using smart search http://smart.embl-heidelberg.de/ .
pr; padr domain; ss, signal sequence; tms, transmembrane segment ; ppd, periplasm domain.
proteins with tms are highlighted in bold
validation of array experiments
real-time rt-pcr was performed on independent samples to validate our array results.
to verify that upregulation of the genes were the result of specific treatment with as- <dig> and not a general response, we also applied samples that were incubated in the presence of sublethal amount of bacitracin  or nisin , bacteriocins that both affect cell wall biosynthesis through blocking the lipid ii cycle by interaction with c55-isoprenyl pyrophosphate  <cit>  or forming pores in cell membrane during interaction with lipid ii  <cit> , respectively.
two genes were selected , both coding for a putative membrane protein and both located downstream of a padr like regulator , for quantitative real time rt-pcr.
quantitative real time pcr showed  <dig> ±  <dig> and  <dig> ±  <dig> times upregulation of bc <dig> and bc <dig> in samples treated with as- <dig> compared with control samples, respectively .
similar analysis of samples treated with as- <dig> for  <dig> min showed less then  <dig> times induction of the bc <dig> and bc <dig> genes, in agreement with the lack of significant changes after  <dig> min of as- <dig> treatment in microarray experiments.
samples incubated in the presence of bacitracin showed slightly enhanced expression of target genes, while addition of nisin did not significantly change the transcription of these genes.
overexpression of bc <dig> increases resistance against as- <dig> in b. cereus and b. subtilis
to resolve whether increased expression of the putative membrane protein bc <dig> helps b. cereus to defend itself against as- <dig>  bc <dig> was cloned behind the iptg inducible pspac promoter and expression was induced in b. cereus atcc <dig>  after preliminary induction of b. cereus containing patk <dig> using  <dig> mm of iptg, cells were exposed to varying amounts of as- <dig> and growth was followed in time.
as depicted in table  <dig>  cells containing overexpressed bc <dig> were able to survive in the presence of slightly increased amounts of as- <dig>  compared to cultures containing control plasmid plm <dig> or when bc <dig> was not induced.
important to note is that bc <dig> is already expressed in wild type b. cereus in response to as- <dig> explaining the relatively low level of increased resistance upon further overexpression of bc <dig>  unfortunately, we were not able to obtain a knockout of bc <dig> to show the expected increased sensitivity.
to support the idea that the increased resistance of b. cereus cells against as- <dig> is caused by specific overexpression of the bc <dig> membrane protein, we randomly selected two membrane proteins  and introduced them into b. cereus atcc <dig> similar to the bc <dig> protein.
expression of these proteins resulted in no significant growth difference in the presence of various amounts of as- <dig> compared to the strain containing the plm <dig> control plasmid.
further, comparative transcriptome analyses of b. cereus carrying plm <dig> control plasmid and the bc <dig> overexpressing plasmid patk <dig> in the presence of iptg revealed the significant  upregulation of the bc <dig> gene  and downregulation of the bc <dig> and bc <dig> genes , when bc <dig> was expressed .
b. cereus containing patk <dig> was challenged with bacitracin and nisin, but expression of bc <dig> did not change the resistance of b. cereus against these bacteriocins .
cells were growth in the absence  or presence  of iptg .
minimal inhibitory concentrations are given in μg/ml of as- <dig>
* p-value <  <dig> ; >  <dig> cultures as determined with student's t-test.
no gene coding for a bc <dig> homologue can be identified in the fully sequenced genome of b. subtilis  <dig>  bc <dig> was introduced and expressed in b. subtilis with a similar method used for b. cereus.
upon induction of bc <dig> the sensitivity of b. subtilis was diminished against as- <dig>  liars was previously reported to respond to cell envelope stress and the target gene liai was highly upregulated by liar in response to the addition of bacitracin or nisin to the medium  <cit> .
to see, whether the presence of as- <dig> had similar effects on the expression of the liai gene, we have assayed the expression form liai promoter in response to the addition of as- <dig>  in contrast to the high upregulation of liai in the presence of bacitracin, we have observed no induction of expression when bacteria were incubated with various amount of as- <dig> .
discussion
in the present study a sublethal as- <dig> concentration was used to detect gene expression differences in b. cereus atcc <dig> that result from interaction of as- <dig> with the cells, but not in response to cell death induced by as- <dig>  we aimed to determine which genes help b. cereus to survive confrontation with as- <dig> and identify possible resistance mechanisms.
while there was very mild change in the growth after  <dig> min.
incubation with a sublethal bacteriocin concentration, at least  <dig> genes were affected significantly .
the observed changes in gene expression were mostly related to up-regulation of membrane associated or periplasmic proteins and downregulation of an operon involved in arginine/ornithine catabolism.
downregulation of argnine/ornithine metabolic genes might be related to the slight difference in growth upon as- <dig> treatment that is not apparent using od measurements.
also, this downregulation might cause a change in local ph at the cell wall in view of the decreased catabolic production of nh <dig> and co <dig>  upregulated genes coded for hypothetical membrane proteins or putative transporters.
the bc4206-bc <dig> operon was most heavily upregulated in b. cereus upon as- <dig> treatment.
bc <dig> is padr type regulator, while bc <dig> is a hypothetical membrane protein with  <dig> transmembrane segments.
members of the padr family are known to have a function in regulating cellular pathways resulting in multidrug resistance, virulence or detoxification  <cit> .
these proteins involved in resistance mechanisms, are generally encoded in the vicinity of the padr genes.
overexpression of the bc <dig> protein in both b. cereus and b. subtilis results in elevated resistance against as- <dig>  upon overexpression of bc <dig>  we have found no other genes to be upregulated , suggesting that increase in bc <dig> expression alone raised the resistance of b. cereus against as- <dig>  interestingly, enhanced resistance upon bc <dig> overexpression was specific to enterocin as- <dig> and not observed in the presence of bacitracin or nisin.
bacitracin and nisin both effect cell wall biosynthesis through blocking the lipid ii cycle  <cit>  and forming pores in the cell membrane during interaction with lipid ii  <cit> , respectively.
this is not the case of enterocin as- <dig>  since the primary action of this antimicrobial peptide, like most other bacteriocins, is the disruption of the cytoplasmic membrane.
in spite of recent advances on genome and transcriptome analysis, there are very few reports on the effects of antimicrobial substances on bacterial gene expression.
recently, martínez et al.
<cit>  reported that lactococcin  <dig>  significantly upregulated the expression of  <dig> genes in lactococcus lactis, most of which encode membrane proteins of unknown function and the two component system  cessr  associated with cell-envelope stress.
lcn <dig> is a non pore-forming bacteriocin that inhibits the synthesis of peptidoglycan at the septum in lactococcus lactis.
moreover, the response of a number of gram-positive bacterial species towards cell wall active antibiotics has been studied recently by using genome-wide transcription analysis  <cit> .
essentially, these reports describe a very complex system involving the concerted action of extracellular sigma factors and two-component systems   <cit> .
liars, the b. subtilis homologue of cessr, was unable to activate liai expression in b. subtilis in response to as- <dig> treatment.
therefore, the effect of as- <dig> on bacterial gene expression clearly differs from the mechanisms described earlier for b. subtilis  <cit> .
the precise way in which bc <dig> responds to the presence of as- <dig> needs to be deciphered by further experimental work, including determining the target genes of bc <dig> and the exact signal sensed by this padr-type regulator.
the structure and function of the bc <dig> membrane protein and its role in the resistance mechanism against as- <dig> is also particularly intriguing and target of our future research.
CONCLUSIONS
b. cereus cells, when treated with bacteriocin as- <dig>  increase the expression of the bc <dig> gene coding for a putative membrane protein.
targeted inactivation of the bc <dig> protein might be useful to increase the effect of as- <dig> on food poisoning b. cereus cells.

 bacillus cereus is a gram positive rod-shaped aerobic, endospore-forming bacterium. strains of b. cereus are widely distributed in the environment, mainly in soil, from where they easily spread to many types of foods, especially of vegetable origin, as well as meat, eggs, milk, and dairy products. this bacterium is one of the leading causes of food poisoning in the developed world. b. cereus causes two types of food-borne intoxications. one type is characterized by nausea and vomiting and abdominal cramps and has an incubation period of to hours. this is the "short-incubation" or emetic form of the disease. the second type is manifested primarily by abdominal cramps and diarrhea with an incubation period of to hours. this type is referred to as the "long-incubation" or diarrheal form of the disease. different strategies may be employed to prevent b. cereus poisoning, like heating food above 75°c before use to kill vegetative cells. however, increasing trends for use of packed foods require new food preservation methods to increase the safety levels against b. cereus. one of the current approaches is the use of antimicrobial peptides such as enterocin as- and other bacteriocins. bacteriocins are small, ribosomally-synthesized antimicrobial peptides synthesized and used by one bacterium as to inhibit growth of similar or closely related bacterial strains. bacteriocins are categorized in several ways, e.g. on basis of the producing strain, common resistance mechanisms, and mechanism of killing. enterocin as- is a broad-spectrum antimicrobial peptide produced by enterococcus faecalis s- belonging to class iii of enterococcal bacteriocins or enterocins. enterocin as- is a 70-residue cyclic peptide with a molecular weight of kda. the crystal structure of enterocin as- has been resolved to Ǻ resolution. it is unique with respect to its natural cyclic structure in which n and c termini are linked by a peptide bond. it has been shown that enterocin as- adopts different oligomeric structures according to physiochemical conditions: it exists in monomeric form at ph below and in dimeric form in the ph range of to. the molecules of as- in the crystal are arranged in chains of pairs of molecules linked either by hydrophobic interactions, or by hydrophilic interactions. the molecules within the df-i interact through the hydrophobic helices h and h on the other hand, the hydrophilic surfaces of helices h and h are interacting in df-ii. the mode of action of enterocin as- has been elucidated. this bacteriocin makes pores of an approximate size of nm in the bacterial cytoplasmic membrane thereby disrupting the proton motive force and causing cell death. it also shows a secondary, bacteriolytic effect against some of the target bacteria. based on its crystal structure, the proposed mechanism of action suggests that the two different stages of molecular association, df-i and df-ii, are involved in changing from the water-soluble df-i to the membrane-bound df-ii stage at the membrane surface. this transition implies a 90° rotation of each protomer within df-i, in a way that the partially hidden hydrophobic helices h and h become solvent accessible. this would permit as- to insert into the bacterial membrane. although the mechanism of action of enterocin as- has been studied extensively at physiological and physico-chemical levels, nothing is known about the responses of sensitive bacterial cells upon exposure to the bacteriocin. previous experiment in our laboratory with as- against listeria monocytogenes showed that bacterial cells can be adapted to as- thereby increasing resistance against as-. this adaptation can be achieved with subsequent inoculation in the presence of low, but still inhibitory concentrations of as- however, the adaptation is gradually lost upon repeated subcultivation. given the great interest of enterocin as- as a food preservative, it is of high relevance to know how the target bacteria react to bacteriocin treatment. this may have direct implications on the elucidation of probable mechanisms for cell adaptation as well as the development of bacteriocin resistance mechanisms. moreover, a better knowledge of the bacterial response to enterocin as- may also allow identification of new targets that could be exploited to enhance bacteriocin activity. the purpose of the present study was to determine the genome-wide response of b. cereus cells exposed to enterocin as- and to identify components that help the bacterium to survive bacteriocin treatments.  effect of enterocin as- on global gene expression in b. cereus atcc14579 enterocin as- was shown to inhibit growth of vegetative cells and spore outgrowth of b. cereus and it can be an effective bioagent against b. cereus in various food related media, e.g. hard cheese, rice based foods, fruit and vegetable juices. although the mode of action of as- is well understood, the response of bacteria to enterocin as- is poorly examined. we have therefore determined the transcriptome of b. cereus atcc in response to as- to omit the effect of growth inhibition related differences between the treated and the control culture, a subinhibitory bacteriocin concentration of μg/ml of as- was used in our experiments. we observed no adaptation process, when b. cereus was subsequently cultivated in the presence of μg/ml of as- but only when cells were treated with low, but inhibitory concentration of as-. in preliminary experiments samples were taken and min after addition of as- but we could not detect any significant changes after min, therefore final transcriptome comparisons were performed on samples incubated with as- for min. transcriptome analyses were performed on six independent biological replicates. genes were identified to be significantly upregulated, and only genes to be downregulated. all downregulated genes are located in one putative operon, coding for proteins involved in arginine and proline metabolism. most of the upregulated genes code for proteins located in the membrane or contain a signal sequence or periplasmic domain. the putative membrane proteins show similarity to different transport or permease proteins or have been annotated as a hypothetical protein with no known function. two regulators belonging to the padr family were significantly upregulated, both located upstream of, and in one operon with, genes coding for membrane proteins that are similarly enhanced during our experiments. here, we selected the most upregulated putative operon for further characterization. the bc4206- operon is conserved in all fully sequenced b. cereus, b thuringiensis and b. weihenstephanensis genomes except in the b. cereus cytotoxis strain, but is missing in b. anthracis and other bacillus species. when this operon is found in a genome, the genes surrounding this operon are also conserved. a the ratio of gene expression is shown. ratio: expression in as- treated sample over that in untreated samples. b bayesian p value c putative function of protein as annotated in the b. cereus atcc genome sequence d domains detected using smart search http://smart.embl-heidelberg.de/. pr; padr domain; ss, signal sequence; tms, transmembrane segment; ppd, periplasm domain. proteins with tms are highlighted in bold validation of array experiments real-time rt-pcr was performed on independent samples to validate our array results. to verify that upregulation of the genes were the result of specific treatment with as- and not a general response, we also applied samples that were incubated in the presence of sublethal amount of bacitracin or nisin, bacteriocins that both affect cell wall biosynthesis through blocking the lipid ii cycle by interaction with c55-isoprenyl pyrophosphate or forming pores in cell membrane during interaction with lipid ii, respectively. two genes were selected, both coding for a putative membrane protein and both located downstream of a padr like regulator, for quantitative real time rt-pcr. quantitative real time pcr showed ± and ± times upregulation of bc and bc in samples treated with as- compared with control samples, respectively. similar analysis of samples treated with as- for min showed less then times induction of the bc and bc genes, in agreement with the lack of significant changes after min of as- treatment in microarray experiments. samples incubated in the presence of bacitracin showed slightly enhanced expression of target genes, while addition of nisin did not significantly change the transcription of these genes. overexpression of bc increases resistance against as- in b. cereus and b. subtilis to resolve whether increased expression of the putative membrane protein bc helps b. cereus to defend itself against as- bc was cloned behind the iptg inducible pspac promoter and expression was induced in b. cereus atcc after preliminary induction of b. cereus containing patk using mm of iptg, cells were exposed to varying amounts of as- and growth was followed in time. as depicted in table cells containing overexpressed bc were able to survive in the presence of slightly increased amounts of as- compared to cultures containing control plasmid plm or when bc was not induced. important to note is that bc is already expressed in wild type b. cereus in response to as- explaining the relatively low level of increased resistance upon further overexpression of bc unfortunately, we were not able to obtain a knockout of bc to show the expected increased sensitivity. to support the idea that the increased resistance of b. cereus cells against as- is caused by specific overexpression of the bc membrane protein, we randomly selected two membrane proteins and introduced them into b. cereus atcc similar to the bc protein. expression of these proteins resulted in no significant growth difference in the presence of various amounts of as- compared to the strain containing the plm control plasmid. further, comparative transcriptome analyses of b. cereus carrying plm control plasmid and the bc overexpressing plasmid patk in the presence of iptg revealed the significant upregulation of the bc gene and downregulation of the bc and bc genes, when bc was expressed. b. cereus containing patk was challenged with bacitracin and nisin, but expression of bc did not change the resistance of b. cereus against these bacteriocins. cells were growth in the absence or presence of iptg. minimal inhibitory concentrations are given in μg/ml of as- * p-value <; > cultures as determined with student's t-test. no gene coding for a bc homologue can be identified in the fully sequenced genome of b. subtilis bc was introduced and expressed in b. subtilis with a similar method used for b. cereus. upon induction of bc the sensitivity of b. subtilis was diminished against as- liars was previously reported to respond to cell envelope stress and the target gene liai was highly upregulated by liar in response to the addition of bacitracin or nisin to the medium. to see, whether the presence of as- had similar effects on the expression of the liai gene, we have assayed the expression form liai promoter in response to the addition of as- in contrast to the high upregulation of liai in the presence of bacitracin, we have observed no induction of expression when bacteria were incubated with various amount of as-. discussion in the present study a sublethal as- concentration was used to detect gene expression differences in b. cereus atcc that result from interaction of as- with the cells, but not in response to cell death induced by as- we aimed to determine which genes help b. cereus to survive confrontation with as- and identify possible resistance mechanisms. while there was very mild change in the growth after min. incubation with a sublethal bacteriocin concentration, at least genes were affected significantly. the observed changes in gene expression were mostly related to up-regulation of membrane associated or periplasmic proteins and downregulation of an operon involved in arginine/ornithine catabolism. downregulation of argnine/ornithine metabolic genes might be related to the slight difference in growth upon as- treatment that is not apparent using od measurements. also, this downregulation might cause a change in local ph at the cell wall in view of the decreased catabolic production of nh and co upregulated genes coded for hypothetical membrane proteins or putative transporters. the bc4206-bc operon was most heavily upregulated in b. cereus upon as- treatment. bc is padr type regulator, while bc is a hypothetical membrane protein with transmembrane segments. members of the padr family are known to have a function in regulating cellular pathways resulting in multidrug resistance, virulence or detoxification. these proteins involved in resistance mechanisms, are generally encoded in the vicinity of the padr genes. overexpression of the bc protein in both b. cereus and b. subtilis results in elevated resistance against as- upon overexpression of bc we have found no other genes to be upregulated, suggesting that increase in bc expression alone raised the resistance of b. cereus against as- interestingly, enhanced resistance upon bc overexpression was specific to enterocin as- and not observed in the presence of bacitracin or nisin. bacitracin and nisin both effect cell wall biosynthesis through blocking the lipid ii cycle and forming pores in the cell membrane during interaction with lipid ii, respectively. this is not the case of enterocin as- since the primary action of this antimicrobial peptide, like most other bacteriocins, is the disruption of the cytoplasmic membrane. in spite of recent advances on genome and transcriptome analysis, there are very few reports on the effects of antimicrobial substances on bacterial gene expression. recently, martínez et al. reported that lactococcin significantly upregulated the expression of genes in lactococcus lactis, most of which encode membrane proteins of unknown function and the two component system cessr associated with cell-envelope stress. lcn is a non pore-forming bacteriocin that inhibits the synthesis of peptidoglycan at the septum in lactococcus lactis. moreover, the response of a number of gram-positive bacterial species towards cell wall active antibiotics has been studied recently by using genome-wide transcription analysis. essentially, these reports describe a very complex system involving the concerted action of extracellular sigma factors and two-component systems. liars, the b. subtilis homologue of cessr, was unable to activate liai expression in b. subtilis in response to as- treatment. therefore, the effect of as- on bacterial gene expression clearly differs from the mechanisms described earlier for b. subtilis. the precise way in which bc responds to the presence of as- needs to be deciphered by further experimental work, including determining the target genes of bc and the exact signal sensed by this padr-type regulator. the structure and function of the bc membrane protein and its role in the resistance mechanism against as- is also particularly intriguing and target of our future research.  b. cereus cells, when treated with bacteriocin as- increase the expression of the bc gene coding for a putative membrane protein. targeted inactivation of the bc protein might be useful to increase the effect of as- on food poisoning b. cereus cells. 
the operon structure and genes are conserved in b. cereus and b. thuringiensis species, but are not present in b. anthracis and b. subtilis. upon overexpression of bc in b. cereus, we observed an increased resistance against as- expression of bc in b. subtilis which lacks this operon also showed increased resistance against as- bc membrane protein is involved in the resistance mechanism of b. cereus cells against as- of the genes analysed, expression of genes was found to change significantly after a min treatment with a subinhibitory bacteriocin concentration of μg/ml. due to its activity against various gram positive and some gram negative bacteria it has clear potential for use as a food preservative. bc codes for a padr type transcriptional regulator, while bc codes for a hypothetical membrane protein. here, we studied the effect of enterocin as- challenges on vegetative cells of bacillus cereus atcc by use of transcriptome analysis.
 enterocin as- is produced by enterococcus faecalis s to compete with other bacteria in their environment. due to its activity against various gram positive and some gram negative bacteria it has clear potential for use as a food preservative. here, we studied the effect of enterocin as- challenges on vegetative cells of bacillus cereus atcc by use of transcriptome analysis.  of the genes analysed, expression of genes was found to change significantly after a min treatment with a subinhibitory bacteriocin concentration of μg/ml. most of up-regulated genes encode membrane-associated or secreted proteins with putative transmembrane segments or signal sequences, respectively. one operon involved in arginine metabolism was significantly downregulated. the bc4206-bc operon was found to be the most upregulated target in our experiments. bc codes for a padr type transcriptional regulator, while bc codes for a hypothetical membrane protein. the operon structure and genes are conserved in b. cereus and b. thuringiensis species, but are not present in b. anthracis and b. subtilis. using real-time qpcr, we show that these genes are upregulated when we treated the cells with as- but not upon nisin treatment. upon overexpression of bc in b. cereus, we observed an increased resistance against as- expression of bc in b. subtilis which lacks this operon also showed increased resistance against as-  bc membrane protein is involved in the resistance mechanism of b. cereus cells against as- 