BACKGROUND
listeria monocytogenes is a food-borne facultative intracellular pathogen that causes a wide spectrum of clinical disease in humans, ranging from mild influenza-like illness and gastroenteritis to severe listeriosis with meningitis, which is frequently accompanied by septicemia and meningoencephalitis.
while listeriosis may occur in otherwise healthy individuals, those primarily at risk are immunocompromised patients, pregnant women, the very young and the elderly  <cit> .
the antibiotics of choice in the treatment of listeriosis are the β-lactams penicillin g and ampicillin, alone or in combination with gentamicin.
however, despite the use of antibiotic therapy, up to one-third of patients die  <cit> .
in general, isolates of l. monocytogenes are susceptible to β-lactam antibiotics, except for members of the cephalosporin family.
however, for most isolates, there is a large gap between the mic  and mbc  values of β-lactam antibiotics.
consequently, l. monocytogenes is regarded as tolerant to all β-lactams  <cit> .
furthermore, the high level of innate resistance of l. monocytogenes to cephalosporins may be especially significant since members of this family of β-lactams are frequently used to treat sepsis of unknown etiology.
tolerance to β-lactams and innate resistance to cephalosporins are among the most important factors contributing to the not infrequent ineffectiveness of antibiotic therapy of listeriosis.
in an effort to decrease the significant human and economic costs associated with listeriosis, the development of methodologies to reduce the survival and growth of l. monocytogenes during infection is the focus of much research effort.
one of the primary goals is to characterize the mechanisms of susceptibility and tolerance of l. monocytogenes to β-lactams.
to date, a number of genes that play a role in the innate resistance of l. monocytogenes to cephalosporins have been identified.
of these, lmo <dig>  lmo <dig> and lmo <dig> encode penicillin binding proteins that are the classical target enzymes for β-lactam antibiotics  <cit> .
other examples of genes contributing to innate resistance are mdrl, which encodes an antibiotic efflux pump  <cit> , tela a gene homologous to tellurite resistance loci  <cit> , anrab, which encodes a putative multidrug resistance transporter  <cit>  and lmo <dig> a homolog of enterococcus faecium vanz <cit> .
in addition, the two-component systems  cesrk and lisrk have been identified as key mediators involved in the innate resistance of l. monocytogenes to cephalosporins  <cit> .
most recently, genome-wide transcriptional studies have confirmed the crucial role of lisrk and cesrk in the response of l. monocytogenes to β-lactams and have demonstrated that two other tcss, liasr and virrs, are also linked to this response  <cit> .
the mechanisms of tolerance of l. monocytogenes to cell envelope-acting antimicrobial agents are much more poorly characterized than the mechanisms of innate resistance to cephalosporins.
to date, only the alternative sigma factor sigb has been shown to determine the tolerance of l. monocytogenes to β-lactams  <cit> .
it seems reasonable to assume that certain genes that are important for the survival and growth of bacteria in the presence of cell envelope-acting antibiotics are induced during treatment with these antimicrobial agents.
several studies have provided evidence to support this assumption in the case of l. monocytogenes.
stack et al.
<cit>  showed that htra, encoding an htra-like serine protease, is essential for the growth of l. monocytogenes in the presence of penicillin g, and that this gene is more efficiently transcribed when this β-lactam is present.
gottschalk et al.
<cit>  demonstrated that the transcription of several cell wall-related genes  is induced by β-lactam and glycopeptide antibiotics.
three of these genes, lmo <dig>  lmo <dig> and lmo <dig>  play a significant role in the survival of the bacterium in the presence of cell wall-acting antibiotics.
more recently, nielsen et al.
<cit>  showed the same relationship between the induction of expression and significance of lmo <dig> and lmo <dig> genes in the susceptibility of l. monocytogenes to the β-lactam antibiotic cefuroxime.
these observations prompted us to attempt to identify l. monocytogenes genes induced in the presence of penicillin g, in order to learn more about mechanisms of tolerance to this class of antibiotic.
for this purpose, a promoter-trap system based on a promoterless plasmid-borne copy of the hly gene encoding listeriolysin o  was employed.
this system has been used previously to identify l. monocytogenes promoters that are either constitutive or specifically induced during in vivo infection  <cit> .
in the course of this study, ten penicillin-g inducible genes were identified.
the upregulated expression of these genes under penicillin g pressure was verified by transcriptional analysis.
three of the identified genes, namely fri, phop and axyr, were selected for further investigation.
the fri gene encodes a non-heme, iron-binding ferritin-like protein  that belongs to the dps  family of proteins, which play important roles in the response to multiple stresses in many bacterial species .
gene phop encodes a two-component phosphate-response regulator homologous to b. subtilis phop, which plays a crucial role in controlling the biosynthesis of teichoic acid, a key component of the gram-positive bacterial cell wall  <cit> .
gene axyr encodes a transcriptional regulator from the arac/xyls family, some members of which are involved in tolerance to antibiotics  <cit> .
the susceptibility and tolerance to β-lactams of nonpolar deletion mutants in the three selected genes was examined.
it was revealed that fri is a mediator of tolerance to penicillin g and ampicillin, as well as of resistance to some cephalosporins, including cefalotin and cephradine.
the identification of a locus that contributes to tolerance to β-lactams used in the treatment of listeriosis and that is relevant to the innate resistance of l. monocytogenes to cephalosporins is notable in light of the clinical use of these antibiotics.
RESULTS
screening of l. monocytogenes genomic libraries for penicillin g-inducible promoters
genomic dna of l. monocytogenes was fragmented using four different procedures and the obtained chromosomal fragments were cloned upstream of the promoterless hly gene in vector pat28-hly.
this vector has previously been used to identify constitutive as well as inducible promoters of l. monocytogenes <cit> .
it was chosen for the identification of penicillin g-inducible promoters because the plasmid is present in l. monocytogenes at high copy number, which permits the selection of even relatively weak promoters driving hly expression.
penicillin g was selected for this study because it is widely used as the antibiotic of choice for the treatment of listerial infections  <cit> .
the four genomic libraries were introduced into l. monocytogenes egdΔhly by electroporation and transformed strains in which putative promoters were trapped upstream of hly, were identified by the creation of hemolytic zones on blood agar plates.
to determine whether expression was induced by penicillin g, the strains were replica plated on blood agar plates with or without this antibiotic.
penicillin g was used at a concentration  that permitted the growth of l. monocytogenes egd even under prolonged incubation, but which exerted a deleterious effect on the bacteria, as evidenced by a reduced growth rate and lower cell number compared with cultures without the antibiotic.
strains producing larger hemolytic zones on blood agar plates supplemented with penicillin g were identified.
inducible expression of the promoter-hly fusions in the selected strains in response to the addition of penicillin g was further quantified using a hemolytic activity assay.
in the presence of penicillin g a significant increase in hemolytic activity produced by nine of the selected strains was observed .
a the hemolytic activity was measured for selected strains carrying promoter-hly fusions grown for 2 h in the presence  or absence  of penicillin g , using b <dig> %, c1% or d2% suspensions of srbc.
the results are the average of three independent experiments, each performed in triplicate ± the standard deviation.
asterisks indicate significant differences according to student’s t test .
analysis of trapped chromosomal dna fragments in strains showing penicillin g-inducible hly expression
the chromosomal fragments carrying penicillin g-inducible promoters were sequenced and compared with the l. monocytogenes egd-e genome.
in the case of seven strains, namely  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> , this analysis identified single genes as the source of the trapped chromosomal dna fragments.
in the case of strain  <dig>  the trapped fragment was comprised of sequences originating from two genes, lmo <dig> and lmo <dig>  both present in the opposite transcriptional orientation to the reporter gene.
it was reasoned that the identified promoter might originate from a divergently transcribed gene positioned immediately upstream of the cloned fragment, but examination of the genome sequence showed that the two preceding genes, lmo <dig> and lmo <dig>  are in the same orientation as lmo <dig> and lmo <dig>  thus, the identified promoter could not direct the expression of any of these genes and for this reason it was excluded from further investigations.
in the case of strain  <dig>  the trapped chromosomal fragment contained the full sequence of genes lmo <dig>  and lmo <dig> plus sequences upstream of these genes, as well as a fragment of the sequence preceding gene lmo <dig>  which is in the same transcriptional orientation.
thus, on the basis of simple sequence analysis it was not possible to identify which promoter was directing hly expression in this strain.
in an attempt to clarify this situation, the possible cotranscription of fri, lmo <dig> and lmo <dig> was examined by rt-pcr.
the three anticipated pcr products were amplified from cdna generated by reverse transcription using primers specific for genes lmo <dig> and lmo <dig>  which demonstrated that fri, lmo <dig> and lmo <dig> are cotranscribed in both non-stressed cells and in cells grown under penicillin g pressure .
consequently, each of these genes was analyzed further.
a nucleotide position in genome of l. monocytogenes egd  given in the same orientation as the reporter gene.
bgenes/fragments of genes and intergenic region present in the trapped fragments, with the sequence located directly upstream of the 5′ end of the hly gene marked in bold, while the genes/fragments of genes in the same orientation as this reporter gene are underlined.
the genes whose promoters were identified as responsible for increased hly expression in the presence of penicillin g were further characterized  and four of them were found to have established functions.
gene phop encodes a transcriptional regulator of the two-component system phopr, fri encodes a non-heme iron-binding ferritin involved in adaptation to atypical conditions, leus encodes a leucyl-trna synthetase engaged in protein synthesis, and axyr encodes a putative transcriptional regulator with homology to arac/xyls regulators.
the functions of the proteins encoded by the six other identified penicillin g-inducible genes are unknown, but some predictions could be made on the basis of their homology to proteins with putative functions and/or the presence of domains possessing a specific function.
proteins lmo <dig> and lmo <dig> possess putative signal sequences and thus could represent surface proteins of l. monocytogenes.
lmo <dig> shows homology to the c-terminal region of the dna binding and competence protein comec as well as comea of b. subtilis .
in the case of the four other putative proteins, three are homologs of proteins in b. subtilis: lmo <dig> exhibits similarity to the yner protein , lmo <dig> shares homology with the yxko protein , and lmo <dig> is homologous to protein yktb .
the other protein, lmo <dig> is highly similar to hypothetical bacterial proteins of unknown function.
a based on data available from the ncbi .
b functions are based on annotations provided by the listilist .
transcriptional analysis of the identified genes in the presence of penicillin g
to verify penicillin g-inducible expression of the identified genes in wild-type l. monocytogenes egd, transcriptional analysis in non-stressed cells and in cells growing under penicillin g pressure was performed, and their relative expression levels were quantified .
this analysis confirmed that the annotated genes downstream of the captured dna in each clone were indeed upregulated in response to the presence of penicillin g, thus validating the results obtained with the hly reporter system.
in addition, the transcriptional analysis also demonstrated that the genes identified on the basis of elevated reporter gene expression in the presence of penicillin g during the stationary phase of growth, were also induced by this antibiotic in the exponential phase of growth.
antimicrobial susceptibility of l. monocytogenes Δfri, Δphop and Δaxyr mutant strains
to investigate whether any of the identified genes play a role in the susceptibility of l. monocytogenes to β-lactams, three of them, namely fri, phop and axyr, were selected for further study.
the Δfri mutant was constructed in a previous study  <cit> , while the Δphop and Δaxyr mutants were created using the temperature-sensitive shuttle vector pmad via double-crossover homologous recombination.
prior to detailed investigations, the growth rates of the mutants and the parent strain in bhi broth at 37°c were compared, but no differences were observed .
to determine whether disruption of the phop, axyr and fri genes affected the susceptibility of l. monocytogenes to penicillin g and ampicillin – the antibiotics of choice for the treatment of listerial infections  <cit>  – mic values were determined for the mutants, as was their ability to grow and survive in the presence of sublethal and lethal concentrations of these β-lactams, respectively.
the absence of phop, axyr or fri expression had no effect on the mics of penicillin g and ampicillin, which were identical for all strains .
however, when the ability of the mutants to grow in a sublethal concentration of penicillin g was examined, the Δphop and Δaxyr mutants were found to grow slightly faster than the wild type, whereas the growth of Δfri was impaired .
the same pattern of growth was observed with a sublethal concentration of ampicillin .
the tolerance assay revealed that the ability of the Δphop and Δaxyr mutants to survive in the presence of a lethal concentration of penicillin g was no different to that of the wild type, whereas the tolerance of Δfri was significantly impaired since viable cells of this mutant strain could not be recovered after prolonged exposure to penicillin g .
the same pattern of tolerance of the strains to ampicillin was observed .
to determine whether phop, axyr or fri play a role in the susceptibility to l. monocytogenes to β-lactams other than penicillin g and ampicillin, the wild-type strain and the three mutants were tested in an antibiotic disk assay with cephalosporin, monobactam and carbapenem disks.
this assay did not reveal any significant alterations in the resistance of l. monocytogenes to these antibiotics caused by the lack of functional phop or axyr genes, but significantly greater zones of growth inhibition were observed for the fri mutant with the antibiotics cefalotin and cephradine .
the mics of these specific cephalosporin antibiotics were then determined for l. monocytogenes egd and the Δfri mutant.
in confirmation of the antibiotic disk assay result, the mic of cefalotin for egd and Δfri was 2 μg/ml and 1 μg/ml, respectively, whereas the mic of cephradine for egd and Δfri was 64 μg/ml and 32 μg/ml, respectively.
thus, interruption of the fri gene caused a 2-fold increase in the sensitivity of l. monocytogenes to these cephalosporins.
discussion
in this study, we attempted to identify penicillin g-inducible genes of l. monocytogenes, some of which might be essential for the survival and growth of this bacterium in the presence of cell wall-acting antibiotics.
a promoter trap system was used to identify nine strains showing significantly increased expression of a reporter gene  in the presence of penicillin g. in one case , the predicted polarity of transcription was opposite to that of hly.
the cloned sequence corresponded to fragments of the genes lmo <dig> and lmo <dig>  both of which are involved in the metabolism of carbohydrates.
a recent study examining the transcription of the entire genome of l. monocytogenes has shown that the identified promoter drives the transcription of a long antisense rna with no known physiological role  <cit> .
analysis of the chromosomal dna fragments trapped in the other strains permitted the identification of ten penicillin g-inducible genes.
increased expression of the identified genes in the presence of penicillin g was further confirmed by transcriptional analysis.
the transcription of seven of the identified genes, namely lmo <dig>  lmo <dig>  lmo <dig>  leus, lmo <dig>  phop and axyr, appeared to be upregulated in response to this stress in a growth phase-independent manner, since they were initially identified in the stationary phase of growth and subsequently their elevated expression was also observed in exponentially growing cells.
on the basis of the initial promoter trap system results it was difficult to determine whether the genes fri, lmo <dig> and lmo <dig>  or only one or two of them, show increased expression under penicillin g pressure in the stationary phase of growth.
however all three of these genes were definitely transcriptionally upregulated in response to this stress in the exponential phase of growth.
the functions of the proteins encoded by six of the identified genes are unknown, but four have established functions.
one of them, fri, encodes a ferritin-like protein which belongs to the dps family.
previously, this listerial ferritin was shown to contribute to virulence and to play a role in protection against multiple stresses  <cit> .
the expression of the fri gene is known to be upregulated in a σb-dependent manner  <cit> .
interestingly, sigb was found to determine the tolerance of l. monocytogenes to cell envelope-acting antimicrobial agents  <cit> , and in a Δfri mutant strain, overexpression of an anti-sigma b factor, rsbw, was observed  <cit> , which strongly suggests possible modulation of sigb activity by ferritin.
gene phop, a member of the phosphate starvation two-component regulatory system phop-phor is involved in the regulation of alkaline phosphatase genes in response to environmental signals.
in b. subtilis, it has been shown that the phop-phor system is also involved in controlling the biosynthesis of teichoic acid, a key component of the cell walls of gram-positive bacteria  <cit> .
more recently, it was found that a lack of phor in l. monocytogenes results in altered tolerance to ethanol stress.
this observation suggests that the listerial phop-phor system is involved in regulating the composition of the cell wall  <cit> .
gene axyr encodes a putative bimodular protein with an n-terminal region containing a conserved hth domain required for transcriptional regulation by arac/xyls regulators at targeted promoters  <cit> .
some members of the arac/xyls family are involved in tolerance to antibiotics and others regulate virulence genes.
it has been hypothesized that axyr regulates the expression of the l. monocytogenes virulence factor inlj during in vivo infection  <cit> , and the contribution of this protein to virulence is in line with the observed upregulation of axyr expression during in vitro infection  <cit> .
taking into account the strong indications of their potential role in the response of l. monocytogenes to β-lactam pressure, these three genes were selected for further study.
analysis of Δaxyr and Δphop mutant strains revealed that the absence of these gene products had no effect on the mic values and ability of l. monocytogenes to survive in the presence of a lethal dose of β-lactams, indicating that these proteins do not play a significant role in the susceptibility and tolerance of this bacterium to these antibiotics.
the only difference between these mutant strains and the wild-type was their slightly faster growth in the presence of sublethal concentrations of penicillin g and ampicillin.
under these conditions, cells normally sense damage to the cell wall and respond by significantly reducing their growth rate.
we assume, therefore, that the regulators phop and axyr are involved in transmitting signals to adjust the rate of growth under these adverse conditions.
the experiments examining the role of listerial ferritin in the sensitivity and tolerance of l. monocytogenes to β-lactams produced interesting results.
the tolerance of the Δfri mutant to penicillin g and ampicillin was found to be dramatically lower than that of the wild-type strain.
the recent study of kohanski et al.
<cit>  indicated that there is a strong correlation between the ability of bacteria to survive antibiotic action and the level of hydroxyl radicals in antibiotic-treated cells.
efficient killing of bacteria was observed for those antibiotics that cause increased cellular production of h2o <dig>  which is the end product of an oxidative damage cellular death pathway involving stimulation of the fenton reaction  <cit> .
on the other hand, dps proteins are iron-binding and storage proteins that protect cells from oxidative damage by removing excess ferrous ions from the cytosol, making them unavailable for participation in the fenton reaction  <cit> .
therefore, it is likely that the impaired β-lactam tolerance of l. monocytogenes lacking the dps protein fri results from its inability to prevent the cellular production of hydroxyl radicals.
this hypothesis is supported by a recent study which showed that a dps protein protects salmonella enterica from the fenton-mediated killing mechanism of bactericidal antibiotics  <cit> .
it is noteworthy that the Δfri mutant strain also exhibited increased sensitivity to some cephalosporins – antibiotics to which l. monocytogenes shows high innate resistance – that are often used as the first choice when treating infections of unknown etiology.
interestingly, transcriptional analysis demonstrated that two of the identified penicillin g-inducible genes, lmo <dig> and lmo <dig>  are cotranscribed with fri. this is the first evidence that a gene encoding a dps protein is transcribed together with downstream genes.
as mentioned above, the role of fri in the stress response and virulence is well established, but the functions of lmo <dig> and lmo <dig> and their potential roles in these processes in l. monocytogenes are currently unknown and will be the subject of future studies.
similarly, further research effort is required in order to clarify the potential role of the other identified penicillin g-inducible genes in tolerance and/or susceptibility of l. monocytogenes to β-lactam antibiotics.
CONCLUSIONS
disease outbreaks caused by l. monocytogenes-contaminated foods and the serious illnesses and fatalities that occur in susceptible individuals highlight the importance of understanding the mechanisms that enable this bacterium to survive antibiotic therapy.
the present study resulted in the identification of ten penicillin g-inducible genes of l. monocytogenes.
in-depth examination of the contribution of three of the identified genes, namely fri, phop and axyr, to the susceptibility and tolerance of l. monocytogenes to β-lactams indicated that the regulators phop and axyr do not play a significant role in these reactions.
however, these proteins are probably involved in transmitting signals to adjust the rate of growth of l. monocytogenes under β-lactam pressure.
the most important finding of this research is that the ferritin-like protein fri contributes to l. monocytogenes tolerance of the β-lactam antibiotics penicillin g and ampicillin – the current drugs of choice for the treatment of listeriosis – as well as to the high innate resistance of this bacterium to some cephalosporins.
it is therefore possible that the functions of fri are essential for the survival of l. monocytogenes in the clinical setting.
in light of the key role of l. monocytogenes fri, both in the response to multiple stresses and during infection in vivo, it may represent an attractive target for the development of improved control and treatment strategies for this important pathogen.

 listeria monocytogenes is a food-borne facultative intracellular pathogen that causes a wide spectrum of clinical disease in humans, ranging from mild influenza-like illness and gastroenteritis to severe listeriosis with meningitis, which is frequently accompanied by septicemia and meningoencephalitis. while listeriosis may occur in otherwise healthy individuals, those primarily at risk are immunocompromised patients, pregnant women, the very young and the elderly. the antibiotics of choice in the treatment of listeriosis are the β-lactams penicillin g and ampicillin, alone or in combination with gentamicin. however, despite the use of antibiotic therapy, up to one-third of patients die. in general, isolates of l. monocytogenes are susceptible to β-lactam antibiotics, except for members of the cephalosporin family. however, for most isolates, there is a large gap between the mic and mbc values of β-lactam antibiotics. consequently, l. monocytogenes is regarded as tolerant to all β-lactams. furthermore, the high level of innate resistance of l. monocytogenes to cephalosporins may be especially significant since members of this family of β-lactams are frequently used to treat sepsis of unknown etiology. tolerance to β-lactams and innate resistance to cephalosporins are among the most important factors contributing to the not infrequent ineffectiveness of antibiotic therapy of listeriosis. in an effort to decrease the significant human and economic costs associated with listeriosis, the development of methodologies to reduce the survival and growth of l. monocytogenes during infection is the focus of much research effort. one of the primary goals is to characterize the mechanisms of susceptibility and tolerance of l. monocytogenes to β-lactams. to date, a number of genes that play a role in the innate resistance of l. monocytogenes to cephalosporins have been identified. of these, lmo lmo and lmo encode penicillin binding proteins that are the classical target enzymes for β-lactam antibiotics. other examples of genes contributing to innate resistance are mdrl, which encodes an antibiotic efflux pump, tela a gene homologous to tellurite resistance loci, anrab, which encodes a putative multidrug resistance transporter and lmo a homolog of enterococcus faecium vanz. in addition, the two-component systems cesrk and lisrk have been identified as key mediators involved in the innate resistance of l. monocytogenes to cephalosporins. most recently, genome-wide transcriptional studies have confirmed the crucial role of lisrk and cesrk in the response of l. monocytogenes to β-lactams and have demonstrated that two other tcss, liasr and virrs, are also linked to this response. the mechanisms of tolerance of l. monocytogenes to cell envelope-acting antimicrobial agents are much more poorly characterized than the mechanisms of innate resistance to cephalosporins. to date, only the alternative sigma factor sigb has been shown to determine the tolerance of l. monocytogenes to β-lactams. it seems reasonable to assume that certain genes that are important for the survival and growth of bacteria in the presence of cell envelope-acting antibiotics are induced during treatment with these antimicrobial agents. several studies have provided evidence to support this assumption in the case of l. monocytogenes. stack et al. showed that htra, encoding an htra-like serine protease, is essential for the growth of l. monocytogenes in the presence of penicillin g, and that this gene is more efficiently transcribed when this β-lactam is present. gottschalk et al. demonstrated that the transcription of several cell wall-related genes is induced by β-lactam and glycopeptide antibiotics. three of these genes, lmo lmo and lmo play a significant role in the survival of the bacterium in the presence of cell wall-acting antibiotics. more recently, nielsen et al. showed the same relationship between the induction of expression and significance of lmo and lmo genes in the susceptibility of l. monocytogenes to the β-lactam antibiotic cefuroxime. these observations prompted us to attempt to identify l. monocytogenes genes induced in the presence of penicillin g, in order to learn more about mechanisms of tolerance to this class of antibiotic. for this purpose, a promoter-trap system based on a promoterless plasmid-borne copy of the hly gene encoding listeriolysin o was employed. this system has been used previously to identify l. monocytogenes promoters that are either constitutive or specifically induced during in vivo infection. in the course of this study, ten penicillin-g inducible genes were identified. the upregulated expression of these genes under penicillin g pressure was verified by transcriptional analysis. three of the identified genes, namely fri, phop and axyr, were selected for further investigation. the fri gene encodes a non-heme, iron-binding ferritin-like protein that belongs to the dps family of proteins, which play important roles in the response to multiple stresses in many bacterial species. gene phop encodes a two-component phosphate-response regulator homologous to b. subtilis phop, which plays a crucial role in controlling the biosynthesis of teichoic acid, a key component of the gram-positive bacterial cell wall. gene axyr encodes a transcriptional regulator from the arac/xyls family, some members of which are involved in tolerance to antibiotics. the susceptibility and tolerance to β-lactams of nonpolar deletion mutants in the three selected genes was examined. it was revealed that fri is a mediator of tolerance to penicillin g and ampicillin, as well as of resistance to some cephalosporins, including cefalotin and cephradine. the identification of a locus that contributes to tolerance to β-lactams used in the treatment of listeriosis and that is relevant to the innate resistance of l. monocytogenes to cephalosporins is notable in light of the clinical use of these antibiotics.  screening of l. monocytogenes genomic libraries for penicillin g-inducible promoters genomic dna of l. monocytogenes was fragmented using four different procedures and the obtained chromosomal fragments were cloned upstream of the promoterless hly gene in vector pat28-hly. this vector has previously been used to identify constitutive as well as inducible promoters of l. monocytogenes. it was chosen for the identification of penicillin g-inducible promoters because the plasmid is present in l. monocytogenes at high copy number, which permits the selection of even relatively weak promoters driving hly expression. penicillin g was selected for this study because it is widely used as the antibiotic of choice for the treatment of listerial infections. the four genomic libraries were introduced into l. monocytogenes egdΔhly by electroporation and transformed strains in which putative promoters were trapped upstream of hly, were identified by the creation of hemolytic zones on blood agar plates. to determine whether expression was induced by penicillin g, the strains were replica plated on blood agar plates with or without this antibiotic. penicillin g was used at a concentration that permitted the growth of l. monocytogenes egd even under prolonged incubation, but which exerted a deleterious effect on the bacteria, as evidenced by a reduced growth rate and lower cell number compared with cultures without the antibiotic. strains producing larger hemolytic zones on blood agar plates supplemented with penicillin g were identified. inducible expression of the promoter-hly fusions in the selected strains in response to the addition of penicillin g was further quantified using a hemolytic activity assay. in the presence of penicillin g a significant increase in hemolytic activity produced by nine of the selected strains was observed. a the hemolytic activity was measured for selected strains carrying promoter-hly fusions grown for 2 h in the presence or absence of penicillin g, using b %, c1% or d2% suspensions of srbc. the results are the average of three independent experiments, each performed in triplicate ± the standard deviation. asterisks indicate significant differences according to student’s t test. analysis of trapped chromosomal dna fragments in strains showing penicillin g-inducible hly expression the chromosomal fragments carrying penicillin g-inducible promoters were sequenced and compared with the l. monocytogenes egd-e genome. in the case of seven strains, namely and, this analysis identified single genes as the source of the trapped chromosomal dna fragments. in the case of strain the trapped fragment was comprised of sequences originating from two genes, lmo and lmo both present in the opposite transcriptional orientation to the reporter gene. it was reasoned that the identified promoter might originate from a divergently transcribed gene positioned immediately upstream of the cloned fragment, but examination of the genome sequence showed that the two preceding genes, lmo and lmo are in the same orientation as lmo and lmo thus, the identified promoter could not direct the expression of any of these genes and for this reason it was excluded from further investigations. in the case of strain the trapped chromosomal fragment contained the full sequence of genes lmo and lmo plus sequences upstream of these genes, as well as a fragment of the sequence preceding gene lmo which is in the same transcriptional orientation. thus, on the basis of simple sequence analysis it was not possible to identify which promoter was directing hly expression in this strain. in an attempt to clarify this situation, the possible cotranscription of fri, lmo and lmo was examined by rt-pcr. the three anticipated pcr products were amplified from cdna generated by reverse transcription using primers specific for genes lmo and lmo which demonstrated that fri, lmo and lmo are cotranscribed in both non-stressed cells and in cells grown under penicillin g pressure. consequently, each of these genes was analyzed further. a nucleotide position in genome of l. monocytogenes egd given in the same orientation as the reporter gene. bgenes/fragments of genes and intergenic region present in the trapped fragments, with the sequence located directly upstream of the 5′ end of the hly gene marked in bold, while the genes/fragments of genes in the same orientation as this reporter gene are underlined. the genes whose promoters were identified as responsible for increased hly expression in the presence of penicillin g were further characterized and four of them were found to have established functions. gene phop encodes a transcriptional regulator of the two-component system phopr, fri encodes a non-heme iron-binding ferritin involved in adaptation to atypical conditions, leus encodes a leucyl-trna synthetase engaged in protein synthesis, and axyr encodes a putative transcriptional regulator with homology to arac/xyls regulators. the functions of the proteins encoded by the six other identified penicillin g-inducible genes are unknown, but some predictions could be made on the basis of their homology to proteins with putative functions and/or the presence of domains possessing a specific function. proteins lmo and lmo possess putative signal sequences and thus could represent surface proteins of l. monocytogenes. lmo shows homology to the c-terminal region of the dna binding and competence protein comec as well as comea of b. subtilis. in the case of the four other putative proteins, three are homologs of proteins in b. subtilis: lmo exhibits similarity to the yner protein, lmo shares homology with the yxko protein, and lmo is homologous to protein yktb. the other protein, lmo is highly similar to hypothetical bacterial proteins of unknown function. a based on data available from the ncbi. b functions are based on annotations provided by the listilist. transcriptional analysis of the identified genes in the presence of penicillin g to verify penicillin g-inducible expression of the identified genes in wild-type l. monocytogenes egd, transcriptional analysis in non-stressed cells and in cells growing under penicillin g pressure was performed, and their relative expression levels were quantified. this analysis confirmed that the annotated genes downstream of the captured dna in each clone were indeed upregulated in response to the presence of penicillin g, thus validating the results obtained with the hly reporter system. in addition, the transcriptional analysis also demonstrated that the genes identified on the basis of elevated reporter gene expression in the presence of penicillin g during the stationary phase of growth, were also induced by this antibiotic in the exponential phase of growth. antimicrobial susceptibility of l. monocytogenes Δfri, Δphop and Δaxyr mutant strains to investigate whether any of the identified genes play a role in the susceptibility of l. monocytogenes to β-lactams, three of them, namely fri, phop and axyr, were selected for further study. the Δfri mutant was constructed in a previous study, while the Δphop and Δaxyr mutants were created using the temperature-sensitive shuttle vector pmad via double-crossover homologous recombination. prior to detailed investigations, the growth rates of the mutants and the parent strain in bhi broth at 37°c were compared, but no differences were observed. to determine whether disruption of the phop, axyr and fri genes affected the susceptibility of l. monocytogenes to penicillin g and ampicillin – the antibiotics of choice for the treatment of listerial infections – mic values were determined for the mutants, as was their ability to grow and survive in the presence of sublethal and lethal concentrations of these β-lactams, respectively. the absence of phop, axyr or fri expression had no effect on the mics of penicillin g and ampicillin, which were identical for all strains. however, when the ability of the mutants to grow in a sublethal concentration of penicillin g was examined, the Δphop and Δaxyr mutants were found to grow slightly faster than the wild type, whereas the growth of Δfri was impaired. the same pattern of growth was observed with a sublethal concentration of ampicillin. the tolerance assay revealed that the ability of the Δphop and Δaxyr mutants to survive in the presence of a lethal concentration of penicillin g was no different to that of the wild type, whereas the tolerance of Δfri was significantly impaired since viable cells of this mutant strain could not be recovered after prolonged exposure to penicillin g. the same pattern of tolerance of the strains to ampicillin was observed. to determine whether phop, axyr or fri play a role in the susceptibility to l. monocytogenes to β-lactams other than penicillin g and ampicillin, the wild-type strain and the three mutants were tested in an antibiotic disk assay with cephalosporin, monobactam and carbapenem disks. this assay did not reveal any significant alterations in the resistance of l. monocytogenes to these antibiotics caused by the lack of functional phop or axyr genes, but significantly greater zones of growth inhibition were observed for the fri mutant with the antibiotics cefalotin and cephradine. the mics of these specific cephalosporin antibiotics were then determined for l. monocytogenes egd and the Δfri mutant. in confirmation of the antibiotic disk assay result, the mic of cefalotin for egd and Δfri was 2 μg/ml and 1 μg/ml, respectively, whereas the mic of cephradine for egd and Δfri was 64 μg/ml and 32 μg/ml, respectively. thus, interruption of the fri gene caused a 2-fold increase in the sensitivity of l. monocytogenes to these cephalosporins. discussion in this study, we attempted to identify penicillin g-inducible genes of l. monocytogenes, some of which might be essential for the survival and growth of this bacterium in the presence of cell wall-acting antibiotics. a promoter trap system was used to identify nine strains showing significantly increased expression of a reporter gene in the presence of penicillin g. in one case, the predicted polarity of transcription was opposite to that of hly. the cloned sequence corresponded to fragments of the genes lmo and lmo both of which are involved in the metabolism of carbohydrates. a recent study examining the transcription of the entire genome of l. monocytogenes has shown that the identified promoter drives the transcription of a long antisense rna with no known physiological role. analysis of the chromosomal dna fragments trapped in the other strains permitted the identification of ten penicillin g-inducible genes. increased expression of the identified genes in the presence of penicillin g was further confirmed by transcriptional analysis. the transcription of seven of the identified genes, namely lmo lmo lmo leus, lmo phop and axyr, appeared to be upregulated in response to this stress in a growth phase-independent manner, since they were initially identified in the stationary phase of growth and subsequently their elevated expression was also observed in exponentially growing cells. on the basis of the initial promoter trap system results it was difficult to determine whether the genes fri, lmo and lmo or only one or two of them, show increased expression under penicillin g pressure in the stationary phase of growth. however all three of these genes were definitely transcriptionally upregulated in response to this stress in the exponential phase of growth. the functions of the proteins encoded by six of the identified genes are unknown, but four have established functions. one of them, fri, encodes a ferritin-like protein which belongs to the dps family. previously, this listerial ferritin was shown to contribute to virulence and to play a role in protection against multiple stresses. the expression of the fri gene is known to be upregulated in a σb-dependent manner. interestingly, sigb was found to determine the tolerance of l. monocytogenes to cell envelope-acting antimicrobial agents, and in a Δfri mutant strain, overexpression of an anti-sigma b factor, rsbw, was observed, which strongly suggests possible modulation of sigb activity by ferritin. gene phop, a member of the phosphate starvation two-component regulatory system phop-phor is involved in the regulation of alkaline phosphatase genes in response to environmental signals. in b. subtilis, it has been shown that the phop-phor system is also involved in controlling the biosynthesis of teichoic acid, a key component of the cell walls of gram-positive bacteria. more recently, it was found that a lack of phor in l. monocytogenes results in altered tolerance to ethanol stress. this observation suggests that the listerial phop-phor system is involved in regulating the composition of the cell wall. gene axyr encodes a putative bimodular protein with an n-terminal region containing a conserved hth domain required for transcriptional regulation by arac/xyls regulators at targeted promoters. some members of the arac/xyls family are involved in tolerance to antibiotics and others regulate virulence genes. it has been hypothesized that axyr regulates the expression of the l. monocytogenes virulence factor inlj during in vivo infection, and the contribution of this protein to virulence is in line with the observed upregulation of axyr expression during in vitro infection. taking into account the strong indications of their potential role in the response of l. monocytogenes to β-lactam pressure, these three genes were selected for further study. analysis of Δaxyr and Δphop mutant strains revealed that the absence of these gene products had no effect on the mic values and ability of l. monocytogenes to survive in the presence of a lethal dose of β-lactams, indicating that these proteins do not play a significant role in the susceptibility and tolerance of this bacterium to these antibiotics. the only difference between these mutant strains and the wild-type was their slightly faster growth in the presence of sublethal concentrations of penicillin g and ampicillin. under these conditions, cells normally sense damage to the cell wall and respond by significantly reducing their growth rate. we assume, therefore, that the regulators phop and axyr are involved in transmitting signals to adjust the rate of growth under these adverse conditions. the experiments examining the role of listerial ferritin in the sensitivity and tolerance of l. monocytogenes to β-lactams produced interesting results. the tolerance of the Δfri mutant to penicillin g and ampicillin was found to be dramatically lower than that of the wild-type strain. the recent study of kohanski et al. indicated that there is a strong correlation between the ability of bacteria to survive antibiotic action and the level of hydroxyl radicals in antibiotic-treated cells. efficient killing of bacteria was observed for those antibiotics that cause increased cellular production of h2o which is the end product of an oxidative damage cellular death pathway involving stimulation of the fenton reaction. on the other hand, dps proteins are iron-binding and storage proteins that protect cells from oxidative damage by removing excess ferrous ions from the cytosol, making them unavailable for participation in the fenton reaction. therefore, it is likely that the impaired β-lactam tolerance of l. monocytogenes lacking the dps protein fri results from its inability to prevent the cellular production of hydroxyl radicals. this hypothesis is supported by a recent study which showed that a dps protein protects salmonella enterica from the fenton-mediated killing mechanism of bactericidal antibiotics. it is noteworthy that the Δfri mutant strain also exhibited increased sensitivity to some cephalosporins – antibiotics to which l. monocytogenes shows high innate resistance – that are often used as the first choice when treating infections of unknown etiology. interestingly, transcriptional analysis demonstrated that two of the identified penicillin g-inducible genes, lmo and lmo are cotranscribed with fri. this is the first evidence that a gene encoding a dps protein is transcribed together with downstream genes. as mentioned above, the role of fri in the stress response and virulence is well established, but the functions of lmo and lmo and their potential roles in these processes in l. monocytogenes are currently unknown and will be the subject of future studies. similarly, further research effort is required in order to clarify the potential role of the other identified penicillin g-inducible genes in tolerance and/or susceptibility of l. monocytogenes to β-lactam antibiotics.  disease outbreaks caused by l. monocytogenes-contaminated foods and the serious illnesses and fatalities that occur in susceptible individuals highlight the importance of understanding the mechanisms that enable this bacterium to survive antibiotic therapy. the present study resulted in the identification of ten penicillin g-inducible genes of l. monocytogenes. in-depth examination of the contribution of three of the identified genes, namely fri, phop and axyr, to the susceptibility and tolerance of l. monocytogenes to β-lactams indicated that the regulators phop and axyr do not play a significant role in these reactions. however, these proteins are probably involved in transmitting signals to adjust the rate of growth of l. monocytogenes under β-lactam pressure. the most important finding of this research is that the ferritin-like protein fri contributes to l. monocytogenes tolerance of the β-lactam antibiotics penicillin g and ampicillin – the current drugs of choice for the treatment of listeriosis – as well as to the high innate resistance of this bacterium to some cephalosporins. it is therefore possible that the functions of fri are essential for the survival of l. monocytogenes in the clinical setting. in light of the key role of l. monocytogenes fri, both in the response to multiple stresses and during infection in vivo, it may represent an attractive target for the development of improved control and treatment strategies for this important pathogen. 
the absence of phop or axyr resulted in more rapid growth of the strains in the presence of sublethal concentration of β-lactams, but had no effect on the mic values or the ability to survive a lethal dose of these antibiotics. a lack of fri also caused a 2-fold increase in the sensitivity of l. monocytogenes to cefalotin and cephradine. the contribution of three of these genes, encoding a ferritin-like protein, a two-component phosphate-response regulator and an arac/xyls family transcriptional regulator, to the susceptibility and tolerance of l. monocytogenes to β-lactams was examined by analysis of nonpolar deletion mutants. the results of reporter system studies, verified by transcriptional analysis, identified ten penicillin g-inducible genes. the hly-based promoter trap system was applied to identify penicillin g-inducible genes of l. monocytogenes. in addition, l. monocytogenes has a high level of innate resistance to the cephalosporin family of β-lactams frequently used to treat sepsis of unknown etiology. the present study has identified fri as an important mediator of β-lactam tolerance and innate resistance to cephalosporins in l. monocytogenes. the β-lactam antibiotics penicillin g and ampicillin are the current drugs of choice for the treatment of listerial infections. phop and axyr are probably involved in transmitting signals to adjust the rate of growth of l. monocytogenes under β-lactam pressure, but these regulators do not play a significant role in susceptibility and tolerance to this class of antibiotics. however, the Δfri strain showed impaired growth in the presence of sublethal concentrations of penicillin g and ampicillin and a significantly reduced ability to survive lethal concentrations of these β-lactams. while isolates of l. monocytogenes are susceptible to these antibiotics, their action is only bacteriostatic and consequently, this bacterium is regarded as tolerant to β-lactams. given the high mortality rate of listeriosis despite rational antibiotic therapy, it is important to identify genes that play a role in the susceptibility and tolerance of l. monocytogenes to β-lactams.
 the food-borne pathogen listeria monocytogenes is the causative agent of listeriosis. the β-lactam antibiotics penicillin g and ampicillin are the current drugs of choice for the treatment of listerial infections. while isolates of l. monocytogenes are susceptible to these antibiotics, their action is only bacteriostatic and consequently, this bacterium is regarded as tolerant to β-lactams. in addition, l. monocytogenes has a high level of innate resistance to the cephalosporin family of β-lactams frequently used to treat sepsis of unknown etiology. given the high mortality rate of listeriosis despite rational antibiotic therapy, it is important to identify genes that play a role in the susceptibility and tolerance of l. monocytogenes to β-lactams.  the hly-based promoter trap system was applied to identify penicillin g-inducible genes of l. monocytogenes. the results of reporter system studies, verified by transcriptional analysis, identified ten penicillin g-inducible genes. the contribution of three of these genes, encoding a ferritin-like protein, a two-component phosphate-response regulator and an arac/xyls family transcriptional regulator, to the susceptibility and tolerance of l. monocytogenes to β-lactams was examined by analysis of nonpolar deletion mutants. the absence of phop or axyr resulted in more rapid growth of the strains in the presence of sublethal concentration of β-lactams, but had no effect on the mic values or the ability to survive a lethal dose of these antibiotics. however, the Δfri strain showed impaired growth in the presence of sublethal concentrations of penicillin g and ampicillin and a significantly reduced ability to survive lethal concentrations of these β-lactams. a lack of fri also caused a 2-fold increase in the sensitivity of l. monocytogenes to cefalotin and cephradine.  the present study has identified fri as an important mediator of β-lactam tolerance and innate resistance to cephalosporins in l. monocytogenes. phop and axyr are probably involved in transmitting signals to adjust the rate of growth of l. monocytogenes under β-lactam pressure, but these regulators do not play a significant role in susceptibility and tolerance to this class of antibiotics. listeria monocytogenestolerance to β-lactamsresistance to cephalosporins 