BACKGROUND
the peptidoglycan structure of staphylococcus aureus is a dynamic, three-dimensional meshwork consisting of multiple layers of glycan strands that are crosslinked through peptide bridges.
it determines the bacterial shape and confers protection against the high internal turgor.
characteristic for the staphylococcal peptidoglycan is the long and flexible pentaglycine interpeptide, which branches off the ε-amino group of the l-lysine of the peptidoglycan stem peptide.
the pentaglycine interpeptide is synthesized in a sequential fashion by the femabx family of nonribosomal peptidyl transferases, which use glycyl-trna as a glycine donor.
while femx  adds the first glycine, fema and femb add gly <dig>  and gly <dig> , respectively  <cit> .
although structurally and functionally related, these factors cannot substitute for one another  <cit> .
growth of mutants with a shortened interpeptide is strongly impaired  <cit> .
they display a massive reduction in cell wall crosslinking, aberrant septum formation, and hypersusceptibility to antibiotics including all β-lactams  <cit> .
in methicillin-resistant s. aureus , methicillin resistance is completely abolished upon inactivation of fema, suggesting that the monoglycine peptidoglycan is a very poor substrate for the native penicillin-binding proteins  as well as for the low affinity pbp2a encoded by meca, which confers resistance to β-lactams.
femx and/or fema were therefore regarded as potential targets for novel antibacterial agents, which could restore β-lactam susceptibility in mrsa  <cit> .
while femx was shown to be essential  <cit> , femab null mutants were postulated to require a secondary, yet uncharacterized compensatory or suppressor mutation chr* to stabilize the cell  <cit> .
the phenotype of a femab null mutant thus reflects not only the consequences of the inactivation of the femab operon, but additionally the effects due to the postulated compensatory mutation.
these compensatory events or adaptations are of potential interest, as they may tell us about the interrelationship between cell wall synthesis and other cellular mechanisms.
by re-introducing the femab wild type allele in cis, the compensatory effects were separated from those due to the femab inactivation.
this allowed us to study the consequences of the adaptation events in the presence of a restored pentaglycine interpeptide synthesis machinery.
RESULTS
phenotypic characterization of the femab+ backcross
the femab null mutant as <dig> derived from the mrsa bb <dig> produces only a monoglycine peptidoglycan interpeptide and shows a poorly crosslinked peptidoglycan, aberrant septum formation, methicillin hypersusceptibility, and a reduced growth rate  <cit> .
back-transduction of the wild type femab allele in cis by selecting for the upstream, co-transducible, silent insertion Ω2000chr::tn <dig>  yielding the backcross strain bb <dig>  restored methicillin resistance, but did not increase the growth rate  <cit> .
therefore, survival of as <dig> was suggested to require a postulated compensatory mutation termed chr*, which was retained in bb <dig>  the mrsa strain bb <dig>  which was obtained by transduction of Ω2000chr::tn <dig> into bb <dig>  represents a wild type control strain isogenic to bb <dig> except for the postulated chr* mutation .
amc, methicillin; em, erythromycin; r, resistant; s, susceptible.
bgrowth in lb broth.
nd, not determined.
compared to the highly enlarged cells of as <dig>  cells of strain bb <dig>  which were again able to produce a pentaglycine interpeptide, regained the same size as those of the wild type strain bb <dig> , suggesting a regular cell separation.
the muropeptide pattern of as <dig> showed a highly increased amount of uncrosslinked monomeric muropeptides at the cost of the oligomeric peaks as described earlier  <cit> .
the wild type muropeptide profile was then re-established in bb <dig> as the characteristic peaks of the dimeric, trimeric, and oligomeric muropeptide fractions were indistinguishable from those of bb <dig> .
however, calculation of the percentage of free reducing termini in the peptidoglycan revealed on average slightly longer glycan chains in as <dig> and bb <dig> than in bb <dig>  as confirmed by two-sided t-test, suggesting that as <dig> may have compensated for the poorly crosslinked cell wall by generating longer sugar chains.
in addition to the reduced growth rate, which may be a further strategy to cope with the cell wall defects caused by the femab deletion, we found that as <dig> and bb <dig> also shared temperature sensitivity .
the autolytic banding patterns and spontaneous and triton x-100-induced autolysis, at both  <dig> and 42°c, were virtually identical in the slowly growing femab+ backcross bb <dig> and the wild type bb <dig> , suggesting that there was no correlation between the autolytic behaviour of bb <dig> and the reduced growth rates observed.
although the overall autolysis did not differ, subtle modulation in autolytic activities may count for the slightly increased glycan chain length.
the strains used in this study all carry a functional meca gene and thus are mrsa.
a characteristic feature of mrsa strains is the heterogeneous expression of resistance to methicillin and other penicillinase-stable β-lactams such as oxacillin, whereby the majority of cells have only a low resistance level.
upon exposure to inhibitory concentrations of β-lactams, a subpopulation with high resistance is selected.
once formed, high resistance is maintained in absence of selective pressure resulting in mrsa with homogeneous oxacillin resistance.
interestingly, detailed analyses showed that the oxacillin resistance of the femab+ backcross bb <dig> had become higher than that of the parental strain bb <dig> and of bb <dig>  as shown in the population analysis profiles  and by growth on an oxacillin gradient plate .
oxacillin resistance was thus overcompensated in bb <dig> and resembled homogeneous resistance as if selected by passage on inhibitory concentrations of oxacillin.
in contrast, bb <dig> remained hypersusceptible to teicoplanin and bacitracin .
the increased susceptibility to teicoplanin, which interestingly did not extend to vancomycin , may indicate changes in the cell membrane with which the lipophilic anchor of teicoplanin interacts  <cit> .
it is therefore likely that the bacitracin and teicoplanin hypersusceptibility of bb <dig> and as <dig> point to changes in the envelope and membrane organisation.
transcriptome analysis
given the multiple phenotypic traits that were not complemented by restoring the pentaglycine interpeptide, changes in the genome transcription profile were expected.
in a snapshot of the transcriptomes of exponentially growing cells by microarray analysis,  <dig> genes were reported to be downregulated in the femab+ backcross strain bb <dig> compared to the wild type strain bb <dig>  and  <dig> genes were reported to be upregulated .
the list of differentially expressed genes was determined using the moderated t-statistics  <cit> , followed by the family-wise error rate -based p-value adjustment according to holm  <cit> , in order to guarantee high confidence in the selected genes.
the distribution of functional classes within the down- and the upregulated genes  is depicted in pie charts for comparison with their occurrence within the total of genes represented on the chip .
among the downregulated genes in the mutant, categories significantly overrepresented as determined by fisher's exact test comprised transport/binding proteins and lipoproteins, protein synthesis, metabolism of lipids, nucleotides and nucleic acids.
taking into account the decreased growth rate of bb <dig>  this finding may in part reflect the differences in growth between the two strains tested, particularly with regard to protein synthesis.
in contrast, the categories that were overrepresented among the upregulated genes, i.e.
metabolism of amino acids and carbohydrates, pathogenic factors, and phage-related functions, may point to changes in metabolism selected by an overall stress response to the original femab deletion.
athe cellular main roles are in accordance with the annotation on the dogan website  <cit> .
boverrepresented functions either among the down- or the upregulated genes are printed in bold and the respective p-values as determined by fisher's exact test are given.
metabolic differences
to uncover metabolic differences between the two strains, we made use of recent systems biology advances: in contrast to an otherwise isolated analysis of single genes, we computationally linked the transcriptional data with a recently developed metabolic network model for s. aureus  <cit>  .
this procedure allowed us to consider the transcriptional differences between the two strains in a metabolic context.
the mapping of transcriptional data onto a metabolic network, which underlies the employed computational algorithm, allows to identify spots  around which significant regulation occurs, and thus assists in carving out metabolism-related insight from the microarray data.
the top scoring reporter metabolites with p-values smaller than  <dig>  are listed in table  <dig> and an overview of pathways in which many of these reporter metabolites occur is given in figure  <dig>
aa, amino acid metabolism; c, carbohydrate metabolism; co, metabolism of cofactors and vitamins; e, energy metabolism; l, lipid metabolism; n, nucleotide metabolism
the arginine-deiminase pathway was found to be upregulated in bb <dig>  this pathway imports extracellular arginine driven by the simultaneous excretion of ornithine via the arginine-ornithine antiporter arcd .
arginine is then converted by the arginine-deiminase  to citrulline, which is further metabolized by the ornithine transcarbamoylase  to ornithine and carbamoyl phosphate.
the carbamate kinase  finally breaks down the latter into ammonia and carbon dioxide yielding one atp.
this pathway is perceived to act as an atp source under anaerobic conditions and in small colony variants  lacking a functional respiratory chain  <cit> , and it is also thought to be an important player in ph homeostasis as it was also found to be expressed in biofilms  <cit> .
furthermore, depletion of arginine by the arginine-deiminase pathway, which inhibits nitric oxide production in the host, and thus both the innate and the adaptive immune responses against microbial infections, may increase staphylococcal virulence  <cit> .
another observation was a reduction in glna expression in the femab+ backcross bb <dig>  as was confirmed by northern blots of the glnra operon .
the glutamine synthetase  glna produces l-glutamine from l-glutamate and ammonia using one atp to drive the reaction.
glutamine plays a central role in nitrogen metabolism and functions as an amino group donor in many biosynthetic pathways, leading to the synthesis of histidine, tryptophan, carbamoyl phosphate, glucosamine-6-phosphate, purines, and pyrimidines.
a mutation in glnr, which has a polar effect on glna, results in a decreased gs activity and in a reduction of the amidation of the id-glutamate of the peptidoglycan stem peptide thereby reducing methicillin resistance  <cit> .
glna downregulation in bb <dig> may reflect an overall reduced requirement for nitrogen due to slowed down growth.
interestingly, another link to nitrogen metabolism was found in the increased amounts of ureb  and uref  transcripts displayed by strain bb <dig>  which was consistent with the derepression of urease production observed in as <dig> and bb <dig> in urea-containing medium .
on the one hand the urease reaction supplies nitrogen and on the other hand it serves the maintenance of the ph value by formation of ammonium.
urease expression is induced during nitrogen-limited growth  <cit> , and upregulation is observed in glna mutants  <cit> , growth in biofilms  <cit> , and under heat shock conditions  <cit> .
a downregulation of numerous members of the pyrimidine operon comprising pyrab, pyrb, pyrc, and pyre as well as of the regulator pyrr was detected in bb <dig>  the products of the pyr operon are involved in the de novo synthesis of pyrimidine nucleotides from bicarbonate and from intermediates of the central carbon metabolism or via salvage of preformed pyrimidine bases and nucleotides present in the medium.
transcription of the pyr operon was verified by dot blot analysis because of the large transcript expected, using a pyrp-specific probe .
a downregulation of the pyr operon was confirmed in early logarithmic growth phase at an optical density at  <dig> nm  of  <dig> , however, at an od <dig> of  <dig>  pyr mrna levels in bb <dig> were the same or even higher than in bb <dig> .
in bacillus subtilis, pyrr controls the expression of the pyr operon by binding to specific sequences of the pyr mrna thereby leading to attenuation of transcription  <cit>  in response to exogenous uracil and to intracellular ump/phosphoribosyl pyrophosphate pools  <cit> .
richardson et al.
noted that the pyr operon is repressed in response to nitrosative stress in s. aureus  <cit> .
the upregulation of the arginine-deiminase pathway and the urease reaction as well as the downregulation of the glutamine synthesis may point to a potential action of nitrogen regulators on the pyr operon.
control of pyrr may be exerted by glnr and tnra  <cit> , since a glnr/tnra consensus sequence  <cit>  with two mismatches was identified  <dig> to  <dig> bp upstream of the coding region of pyrr in the public staphylococcal genomes.
while according to the microarray data a reduced transcription of pyrr was expected, signals obtained in northern blots using a pyrr-specific probe were stronger in bb <dig> than in bb <dig> .
these were the only discrepancies between microarray data and northern blots found in the open reading frames tested, leaving the regulation of the pyrimidine operon and the link to the regulation of nitrogen-related functions open.
cell wall
although the femab+ backcross strain bb <dig> produced on average slightly longer glycan chains, the transcription of pbpb, coding for the bifunctional pbp <dig> with transglycosylase activity  <cit> , was not detected to be upregulated, as could have been expected, but downregulated.
furthermore, expression of the soluble glycosyltransferase genes, sgta and sgtb  <cit>  and of genes coding for glucosaminidases, which may contribute to an increased glycan chain length, could not be detected to be altered.
these findings do not rule out a posttranscriptional control of autolytic activities by proteases  <cit> , since three genes encoding proteases  were significantly upregulated.
in addition, the decreased expression of dlta, dltb, and dltd in bb <dig> could also contribute to a lower autolytic activity due to a reduced d-alanine esterification of the teichoic acids  <cit> .
membrane and transporters
one of the major components of the membrane, lysylphosphatidylglycerol, is a product of the lysylphosphatidylglycerol synthase fmtc , which adds a positively charged lysyl residue to phosphatidylglycerol  <cit> .
the fmtc/mprf gene, which belongs to the so-called fem and aux genes, and the inactivation of which reduces methicillin and bacitracin resistance  <cit> , was found to be downregulated in bb <dig>  reduced fmtc transcription may on the one hand mirror the reduced growth rate and could on the other hand, by its influence on the membrane charge, also be one of the causes for bacitracin and teicoplanin hypersusceptibility.
one fifth of the genes found differentially regulated in the femab+ backcross bb <dig> code for membrane-associated proteins, mainly transporters and permeases, with the majority showing a reduced transcription level.
interestingly, opucc, which encodes the substrate-binding protein of a glycine betaine/carnitine/choline abc transporter, was upregulated as could be confirmed by northern hybridization showing a stronger transcription of the whole opuc operon in bb <dig> than in bb <dig> .
the uptake of compatible solutes such as glycine betaine, choline and proline is important in osmotic stress response  <cit> , and the upregulation of opuc may indicate an attempt of the femab deletion mutant to balance osmotic pressure due to the weakened cell wall.
in contrast, opud and its homologue, sa <dig>  members of the betaine/carnitine/choline transporter  family, were downregulated in bb <dig>  this latter family of transporters, however, may respond to other kinds of osmotic stress than the opuc operon.
besides quaternary amines or amino acids, solutes such as polyols  and sugars  may play a role in osmoprotection  <cit> .
indeed, genes encoding transporters specific for these classes of compounds were also found to be upregulated in bb1305: namely the glycerol uptake facilitator , the sucrose-specific iibc component of the phosphotransferase system  , and a hypothetical protein similar to scra .
other genes encoding sugar transporters were also induced in bb <dig>  i.e.
sa0208/sa <dig> , sa <dig> , and sa0318/sa <dig> .
considering the observed upregulation of glpf, it is noteworthy that glpd , glpq , and sa <dig>  also appeared to be upregulated in bb <dig>  these may support osmoprotection as glpq catalyzes the conversion of sn-glycero-3-phosphocholine to glycerol-3-phosphate and choline, the latter of which is oxidized to glycine betaine, which is not metabolized further in osmotically stressed s. aureus  <cit> .
stress response and virulence factors
given the temperature sensitivity of bb <dig>  a connection to the heat shock regulon was conceivable.
in fact, the gene encoding the chaperone dnak was downregulated according to the microarray data.
dnak belongs to the hrca and ctsr controlled heat shock regulon in s. aureus  <cit> .
a dnak-specific probe, revealed a  <dig>  kb-transcript in northern blot analysis, most probably covering the hrca operon, and confirmed reduced transcription levels in bb <dig> at 37°c .
unfavourable environmental conditions are known to induce lysogenic phages, and the upregulation of phage-related genes in bb <dig> is most likely to be regarded in the context of a stress response which occurred in the femab deletion mutant as <dig>  various stress conditions have also been described to trigger the expression of virulence factors  <cit> .
in bb <dig>  the transcriptional changes observed included the upregulation of a whole series of virulence genes, such as those coding for lipase , urease ,α-hemolysin precursor , truncated β-hemolysin , serine proteases , cysteine protease  and aureolysin .
since the expression of virulence factors depends on a complex regulatory network, this finding prompted us to analyze the transcription profiles of the major global regulators including sara , sae  and the agr  operon by northern blot during growth.
in accordance with the expression pattern of two representative virulence factors, namely the α-hemolysin precursor  and the serine proteases , the regulators and especially rnaiii and saers peaked at a lower od <dig> in the femab+ backcross bb <dig> than in bb <dig>  at the transition to stationary phase, at an od <dig> of  <dig>  they had already become clearly weaker in the mutant than in the wild type  revealing a remarkably altered timing in the transcription of the main global regulators in bb <dig>
CONCLUSIONS
this is the first thorough characterization of compensatory effects triggered by a shortened pentaglycine interpeptide, the low ability of pbp to crosslink this altered peptidoglycan, and selection for survival.
the poorly crosslinked cell wall may be not strong enough to withstand the cells' high internal turgor.
this imbalance must have been sensed by the cells, which found a way to counteract osmotic stress, though at the cost of a decreased growth rate and temperature sensitivity.
the rearrangements required also involved changes in the expression of metabolic pathways, especially of the arginine-deiminase pathway and the nitrogen metabolism, and seem to be maintained in a stable manner, since they persisted after restoration of the pentaglycine interpeptide.
this demonstrates the vast extent of the compensatory adaptations.
such compensatory adaptations following mutagenesis may happen much more frequently than anticipated, and may be the cause of the often observed experimental inability to fully complement mutations with the original wild type alleles.
cell wall-deficient forms  of s. aureus are able to survive without an intact murein sacculus, and to internalize and persist in macrophages  <cit> .
adaptive responses to l-forms of s. aureus have recently been described by fuller et al.
<cit> , who selected cell wall-deficient mutants with subinhibitory concentrations of penicillin in the presence of elevated osmolality.
similar to what we observed, namely that the reconstituted strain bb <dig> displayed a higher, more homogeneous oxacillin resistance than the wild type, recovery of the cell wall by the l-forms resulted in a stably inherited penicillin resistance that was independent of a β-lactamase or meca.
apparently, the expected negative effect of reduced fmtc or glna expression on oxacillin resistance was compensated in bb <dig>  a phenomenon, that was also observed with other fem, aux or fmt mrsa mutants which still harboured their original mutations but regained resistance by compensatory events when grown in the presence of β-lactams  <cit> .
the temperature sensitivity and the increased susceptibility to bacitracin and teicoplanin are indications for permanent alterations in the membrane.
membrane defects may also be the cause for the premature upregulation of global regulators which in turn most probably triggered the enhanced expression of virulence factors  <cit> .
it is likely that the adaptations represent just one of many ways that s. aureus could respond to femab inactivation.
some of the findings may be secondary effects caused by slow growth and changes within the cell, not contributing to survival, similar to the upregulation of the purine operon in vancomycin intermediate resistant s. aureus  <cit> , that did not contribute to increased glycopeptide resistance  <cit> .
at this stage, we cannot confirm that the numerous adaptive events that occurred in the femab deletion mutant and that are reflected in the femab+ cis-complemented strain were indeed due to mutation.
the phenotypic adaptations revealed here, such as temperature sensitivity, slowed down growth, but no changes in autolytic activities, were strikingly similar to the phenotype caused by mutations in fema after selection for lysostaphin resistant clones  <cit> , which might be evidence for a common survival strategy.
s. aureus has shown here a remarkable ability to compensate and survive a severe condition, such as femab inactivation, which prevents peptidoglycan crosslinking.
implementation of potential femab inhibitors may therefore entail selection of resistant subpopulations having unwanted characteristics, making a combined treatment with other antibiotics highly advisable.

 the peptidoglycan structure of staphylococcus aureus is a dynamic, three-dimensional meshwork consisting of multiple layers of glycan strands that are crosslinked through peptide bridges. it determines the bacterial shape and confers protection against the high internal turgor. characteristic for the staphylococcal peptidoglycan is the long and flexible pentaglycine interpeptide, which branches off the ε-amino group of the l-lysine of the peptidoglycan stem peptide. the pentaglycine interpeptide is synthesized in a sequential fashion by the femabx family of nonribosomal peptidyl transferases, which use glycyl-trna as a glycine donor. while femx adds the first glycine, fema and femb add gly and gly, respectively. although structurally and functionally related, these factors cannot substitute for one another. growth of mutants with a shortened interpeptide is strongly impaired. they display a massive reduction in cell wall crosslinking, aberrant septum formation, and hypersusceptibility to antibiotics including all β-lactams. in methicillin-resistant s. aureus, methicillin resistance is completely abolished upon inactivation of fema, suggesting that the monoglycine peptidoglycan is a very poor substrate for the native penicillin-binding proteins as well as for the low affinity pbp2a encoded by meca, which confers resistance to β-lactams. femx and/or fema were therefore regarded as potential targets for novel antibacterial agents, which could restore β-lactam susceptibility in mrsa. while femx was shown to be essential, femab null mutants were postulated to require a secondary, yet uncharacterized compensatory or suppressor mutation chr* to stabilize the cell. the phenotype of a femab null mutant thus reflects not only the consequences of the inactivation of the femab operon, but additionally the effects due to the postulated compensatory mutation. these compensatory events or adaptations are of potential interest, as they may tell us about the interrelationship between cell wall synthesis and other cellular mechanisms. by re-introducing the femab wild type allele in cis, the compensatory effects were separated from those due to the femab inactivation. this allowed us to study the consequences of the adaptation events in the presence of a restored pentaglycine interpeptide synthesis machinery.  phenotypic characterization of the femab+ backcross the femab null mutant as derived from the mrsa bb produces only a monoglycine peptidoglycan interpeptide and shows a poorly crosslinked peptidoglycan, aberrant septum formation, methicillin hypersusceptibility, and a reduced growth rate. back-transduction of the wild type femab allele in cis by selecting for the upstream, co-transducible, silent insertion Ω2000chr::tn yielding the backcross strain bb restored methicillin resistance, but did not increase the growth rate. therefore, survival of as was suggested to require a postulated compensatory mutation termed chr*, which was retained in bb the mrsa strain bb which was obtained by transduction of Ω2000chr::tn into bb represents a wild type control strain isogenic to bb except for the postulated chr* mutation. amc, methicillin; em, erythromycin; r, resistant; s, susceptible. bgrowth in lb broth. nd, not determined. compared to the highly enlarged cells of as cells of strain bb which were again able to produce a pentaglycine interpeptide, regained the same size as those of the wild type strain bb, suggesting a regular cell separation. the muropeptide pattern of as showed a highly increased amount of uncrosslinked monomeric muropeptides at the cost of the oligomeric peaks as described earlier. the wild type muropeptide profile was then re-established in bb as the characteristic peaks of the dimeric, trimeric, and oligomeric muropeptide fractions were indistinguishable from those of bb. however, calculation of the percentage of free reducing termini in the peptidoglycan revealed on average slightly longer glycan chains in as and bb than in bb as confirmed by two-sided t-test, suggesting that as may have compensated for the poorly crosslinked cell wall by generating longer sugar chains. in addition to the reduced growth rate, which may be a further strategy to cope with the cell wall defects caused by the femab deletion, we found that as and bb also shared temperature sensitivity. the autolytic banding patterns and spontaneous and triton x-100-induced autolysis, at both and 42°c, were virtually identical in the slowly growing femab+ backcross bb and the wild type bb, suggesting that there was no correlation between the autolytic behaviour of bb and the reduced growth rates observed. although the overall autolysis did not differ, subtle modulation in autolytic activities may count for the slightly increased glycan chain length. the strains used in this study all carry a functional meca gene and thus are mrsa. a characteristic feature of mrsa strains is the heterogeneous expression of resistance to methicillin and other penicillinase-stable β-lactams such as oxacillin, whereby the majority of cells have only a low resistance level. upon exposure to inhibitory concentrations of β-lactams, a subpopulation with high resistance is selected. once formed, high resistance is maintained in absence of selective pressure resulting in mrsa with homogeneous oxacillin resistance. interestingly, detailed analyses showed that the oxacillin resistance of the femab+ backcross bb had become higher than that of the parental strain bb and of bb as shown in the population analysis profiles and by growth on an oxacillin gradient plate. oxacillin resistance was thus overcompensated in bb and resembled homogeneous resistance as if selected by passage on inhibitory concentrations of oxacillin. in contrast, bb remained hypersusceptible to teicoplanin and bacitracin. the increased susceptibility to teicoplanin, which interestingly did not extend to vancomycin, may indicate changes in the cell membrane with which the lipophilic anchor of teicoplanin interacts. it is therefore likely that the bacitracin and teicoplanin hypersusceptibility of bb and as point to changes in the envelope and membrane organisation. transcriptome analysis given the multiple phenotypic traits that were not complemented by restoring the pentaglycine interpeptide, changes in the genome transcription profile were expected. in a snapshot of the transcriptomes of exponentially growing cells by microarray analysis, genes were reported to be downregulated in the femab+ backcross strain bb compared to the wild type strain bb and genes were reported to be upregulated. the list of differentially expressed genes was determined using the moderated t-statistics, followed by the family-wise error rate -based p-value adjustment according to holm, in order to guarantee high confidence in the selected genes. the distribution of functional classes within the down- and the upregulated genes is depicted in pie charts for comparison with their occurrence within the total of genes represented on the chip. among the downregulated genes in the mutant, categories significantly overrepresented as determined by fisher's exact test comprised transport/binding proteins and lipoproteins, protein synthesis, metabolism of lipids, nucleotides and nucleic acids. taking into account the decreased growth rate of bb this finding may in part reflect the differences in growth between the two strains tested, particularly with regard to protein synthesis. in contrast, the categories that were overrepresented among the upregulated genes, i.e. metabolism of amino acids and carbohydrates, pathogenic factors, and phage-related functions, may point to changes in metabolism selected by an overall stress response to the original femab deletion. athe cellular main roles are in accordance with the annotation on the dogan website. boverrepresented functions either among the down- or the upregulated genes are printed in bold and the respective p-values as determined by fisher's exact test are given. metabolic differences to uncover metabolic differences between the two strains, we made use of recent systems biology advances: in contrast to an otherwise isolated analysis of single genes, we computationally linked the transcriptional data with a recently developed metabolic network model for s. aureus. this procedure allowed us to consider the transcriptional differences between the two strains in a metabolic context. the mapping of transcriptional data onto a metabolic network, which underlies the employed computational algorithm, allows to identify spots around which significant regulation occurs, and thus assists in carving out metabolism-related insight from the microarray data. the top scoring reporter metabolites with p-values smaller than are listed in table and an overview of pathways in which many of these reporter metabolites occur is given in figure aa, amino acid metabolism; c, carbohydrate metabolism; co, metabolism of cofactors and vitamins; e, energy metabolism; l, lipid metabolism; n, nucleotide metabolism the arginine-deiminase pathway was found to be upregulated in bb this pathway imports extracellular arginine driven by the simultaneous excretion of ornithine via the arginine-ornithine antiporter arcd. arginine is then converted by the arginine-deiminase to citrulline, which is further metabolized by the ornithine transcarbamoylase to ornithine and carbamoyl phosphate. the carbamate kinase finally breaks down the latter into ammonia and carbon dioxide yielding one atp. this pathway is perceived to act as an atp source under anaerobic conditions and in small colony variants lacking a functional respiratory chain, and it is also thought to be an important player in ph homeostasis as it was also found to be expressed in biofilms. furthermore, depletion of arginine by the arginine-deiminase pathway, which inhibits nitric oxide production in the host, and thus both the innate and the adaptive immune responses against microbial infections, may increase staphylococcal virulence. another observation was a reduction in glna expression in the femab+ backcross bb as was confirmed by northern blots of the glnra operon. the glutamine synthetase glna produces l-glutamine from l-glutamate and ammonia using one atp to drive the reaction. glutamine plays a central role in nitrogen metabolism and functions as an amino group donor in many biosynthetic pathways, leading to the synthesis of histidine, tryptophan, carbamoyl phosphate, glucosamine-6-phosphate, purines, and pyrimidines. a mutation in glnr, which has a polar effect on glna, results in a decreased gs activity and in a reduction of the amidation of the id-glutamate of the peptidoglycan stem peptide thereby reducing methicillin resistance. glna downregulation in bb may reflect an overall reduced requirement for nitrogen due to slowed down growth. interestingly, another link to nitrogen metabolism was found in the increased amounts of ureb and uref transcripts displayed by strain bb which was consistent with the derepression of urease production observed in as and bb in urea-containing medium. on the one hand the urease reaction supplies nitrogen and on the other hand it serves the maintenance of the ph value by formation of ammonium. urease expression is induced during nitrogen-limited growth, and upregulation is observed in glna mutants, growth in biofilms, and under heat shock conditions. a downregulation of numerous members of the pyrimidine operon comprising pyrab, pyrb, pyrc, and pyre as well as of the regulator pyrr was detected in bb the products of the pyr operon are involved in the de novo synthesis of pyrimidine nucleotides from bicarbonate and from intermediates of the central carbon metabolism or via salvage of preformed pyrimidine bases and nucleotides present in the medium. transcription of the pyr operon was verified by dot blot analysis because of the large transcript expected, using a pyrp-specific probe. a downregulation of the pyr operon was confirmed in early logarithmic growth phase at an optical density at nm of, however, at an od of pyr mrna levels in bb were the same or even higher than in bb. in bacillus subtilis, pyrr controls the expression of the pyr operon by binding to specific sequences of the pyr mrna thereby leading to attenuation of transcription in response to exogenous uracil and to intracellular ump/phosphoribosyl pyrophosphate pools. richardson et al. noted that the pyr operon is repressed in response to nitrosative stress in s. aureus. the upregulation of the arginine-deiminase pathway and the urease reaction as well as the downregulation of the glutamine synthesis may point to a potential action of nitrogen regulators on the pyr operon. control of pyrr may be exerted by glnr and tnra, since a glnr/tnra consensus sequence with two mismatches was identified to bp upstream of the coding region of pyrr in the public staphylococcal genomes. while according to the microarray data a reduced transcription of pyrr was expected, signals obtained in northern blots using a pyrr-specific probe were stronger in bb than in bb. these were the only discrepancies between microarray data and northern blots found in the open reading frames tested, leaving the regulation of the pyrimidine operon and the link to the regulation of nitrogen-related functions open. cell wall although the femab+ backcross strain bb produced on average slightly longer glycan chains, the transcription of pbpb, coding for the bifunctional pbp with transglycosylase activity, was not detected to be upregulated, as could have been expected, but downregulated. furthermore, expression of the soluble glycosyltransferase genes, sgta and sgtb and of genes coding for glucosaminidases, which may contribute to an increased glycan chain length, could not be detected to be altered. these findings do not rule out a posttranscriptional control of autolytic activities by proteases, since three genes encoding proteases were significantly upregulated. in addition, the decreased expression of dlta, dltb, and dltd in bb could also contribute to a lower autolytic activity due to a reduced d-alanine esterification of the teichoic acids. membrane and transporters one of the major components of the membrane, lysylphosphatidylglycerol, is a product of the lysylphosphatidylglycerol synthase fmtc, which adds a positively charged lysyl residue to phosphatidylglycerol. the fmtc/mprf gene, which belongs to the so-called fem and aux genes, and the inactivation of which reduces methicillin and bacitracin resistance, was found to be downregulated in bb reduced fmtc transcription may on the one hand mirror the reduced growth rate and could on the other hand, by its influence on the membrane charge, also be one of the causes for bacitracin and teicoplanin hypersusceptibility. one fifth of the genes found differentially regulated in the femab+ backcross bb code for membrane-associated proteins, mainly transporters and permeases, with the majority showing a reduced transcription level. interestingly, opucc, which encodes the substrate-binding protein of a glycine betaine/carnitine/choline abc transporter, was upregulated as could be confirmed by northern hybridization showing a stronger transcription of the whole opuc operon in bb than in bb. the uptake of compatible solutes such as glycine betaine, choline and proline is important in osmotic stress response, and the upregulation of opuc may indicate an attempt of the femab deletion mutant to balance osmotic pressure due to the weakened cell wall. in contrast, opud and its homologue, sa members of the betaine/carnitine/choline transporter family, were downregulated in bb this latter family of transporters, however, may respond to other kinds of osmotic stress than the opuc operon. besides quaternary amines or amino acids, solutes such as polyols and sugars may play a role in osmoprotection. indeed, genes encoding transporters specific for these classes of compounds were also found to be upregulated in bb1305: namely the glycerol uptake facilitator, the sucrose-specific iibc component of the phosphotransferase system, and a hypothetical protein similar to scra. other genes encoding sugar transporters were also induced in bb i.e. sa0208/sa, sa, and sa0318/sa. considering the observed upregulation of glpf, it is noteworthy that glpd, glpq, and sa also appeared to be upregulated in bb these may support osmoprotection as glpq catalyzes the conversion of sn-glycero-3-phosphocholine to glycerol-3-phosphate and choline, the latter of which is oxidized to glycine betaine, which is not metabolized further in osmotically stressed s. aureus. stress response and virulence factors given the temperature sensitivity of bb a connection to the heat shock regulon was conceivable. in fact, the gene encoding the chaperone dnak was downregulated according to the microarray data. dnak belongs to the hrca and ctsr controlled heat shock regulon in s. aureus. a dnak-specific probe, revealed a kb-transcript in northern blot analysis, most probably covering the hrca operon, and confirmed reduced transcription levels in bb at 37°c. unfavourable environmental conditions are known to induce lysogenic phages, and the upregulation of phage-related genes in bb is most likely to be regarded in the context of a stress response which occurred in the femab deletion mutant as various stress conditions have also been described to trigger the expression of virulence factors. in bb the transcriptional changes observed included the upregulation of a whole series of virulence genes, such as those coding for lipase, urease,α-hemolysin precursor, truncated β-hemolysin, serine proteases, cysteine protease and aureolysin. since the expression of virulence factors depends on a complex regulatory network, this finding prompted us to analyze the transcription profiles of the major global regulators including sara, sae and the agr operon by northern blot during growth. in accordance with the expression pattern of two representative virulence factors, namely the α-hemolysin precursor and the serine proteases, the regulators and especially rnaiii and saers peaked at a lower od in the femab+ backcross bb than in bb at the transition to stationary phase, at an od of they had already become clearly weaker in the mutant than in the wild type revealing a remarkably altered timing in the transcription of the main global regulators in bb  this is the first thorough characterization of compensatory effects triggered by a shortened pentaglycine interpeptide, the low ability of pbp to crosslink this altered peptidoglycan, and selection for survival. the poorly crosslinked cell wall may be not strong enough to withstand the cells' high internal turgor. this imbalance must have been sensed by the cells, which found a way to counteract osmotic stress, though at the cost of a decreased growth rate and temperature sensitivity. the rearrangements required also involved changes in the expression of metabolic pathways, especially of the arginine-deiminase pathway and the nitrogen metabolism, and seem to be maintained in a stable manner, since they persisted after restoration of the pentaglycine interpeptide. this demonstrates the vast extent of the compensatory adaptations. such compensatory adaptations following mutagenesis may happen much more frequently than anticipated, and may be the cause of the often observed experimental inability to fully complement mutations with the original wild type alleles. cell wall-deficient forms of s. aureus are able to survive without an intact murein sacculus, and to internalize and persist in macrophages. adaptive responses to l-forms of s. aureus have recently been described by fuller et al., who selected cell wall-deficient mutants with subinhibitory concentrations of penicillin in the presence of elevated osmolality. similar to what we observed, namely that the reconstituted strain bb displayed a higher, more homogeneous oxacillin resistance than the wild type, recovery of the cell wall by the l-forms resulted in a stably inherited penicillin resistance that was independent of a β-lactamase or meca. apparently, the expected negative effect of reduced fmtc or glna expression on oxacillin resistance was compensated in bb a phenomenon, that was also observed with other fem, aux or fmt mrsa mutants which still harboured their original mutations but regained resistance by compensatory events when grown in the presence of β-lactams. the temperature sensitivity and the increased susceptibility to bacitracin and teicoplanin are indications for permanent alterations in the membrane. membrane defects may also be the cause for the premature upregulation of global regulators which in turn most probably triggered the enhanced expression of virulence factors. it is likely that the adaptations represent just one of many ways that s. aureus could respond to femab inactivation. some of the findings may be secondary effects caused by slow growth and changes within the cell, not contributing to survival, similar to the upregulation of the purine operon in vancomycin intermediate resistant s. aureus, that did not contribute to increased glycopeptide resistance. at this stage, we cannot confirm that the numerous adaptive events that occurred in the femab deletion mutant and that are reflected in the femab+ cis-complemented strain were indeed due to mutation. the phenotypic adaptations revealed here, such as temperature sensitivity, slowed down growth, but no changes in autolytic activities, were strikingly similar to the phenotype caused by mutations in fema after selection for lysostaphin resistant clones, which might be evidence for a common survival strategy. s. aureus has shown here a remarkable ability to compensate and survive a severe condition, such as femab inactivation, which prevents peptidoglycan crosslinking. implementation of potential femab inhibitors may therefore entail selection of resistant subpopulations having unwanted characteristics, making a combined treatment with other antibiotics highly advisable. 
in addition to slow growth, the cis-complemented femab mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for atp production. a number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. this study characterizes the adaptations that ensured survival of the cells after femab inactivation. in addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sara, and agr. femab mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making femab a potential target to restore β-lactam susceptibility in methicillin-resistant s. aureus. cis-complementation with wild type femab only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of s. aureus to circumvent cell wall damage. potential femab inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors. synthesis of the staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases femx, fema and femb.
 synthesis of the staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases femx, fema and femb. inactivation of the femab operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femab mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making femab a potential target to restore β-lactam susceptibility in methicillin-resistant s. aureus. cis-complementation with wild type femab only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. this study characterizes the adaptations that ensured survival of the cells after femab inactivation.  in addition to slow growth, the cis-complemented femab mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. a number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for atp production. in addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sara, and agr.  survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of s. aureus to circumvent cell wall damage. potential femab inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors. 