BACKGROUND
staphylococcus epidermidis is an opportunistic pathogen which normally inhabits human skin and mucous membranes, primarily infecting immunocompromised individuals or those with implanted biomaterials.
the pathogenicity of s. epidermidis is mostly due to its ability to form a thick, multilayered biofilm on polymeric surfaces  <cit> .
treatment of s. epidermidis infection has become a troublesome problem as biofilm-associated bacteria exhibit enhanced resistance to antibiotics and to components of the innate host defences  <cit> .
among the staphylococci, the other major human pathogen is staphylococcus aureus, which causes infections ranging from cutaneous infections and food poisoning to life-threatening septicaemia.
aside from biofilm, s. aureus produce a large array of exotoxins and exoezymes  <cit> .
two-component regulatory systems  play a pivotal role in bacterial adaptation, survival, and virulence by sensing changes in the external environment and modulating gene expression in response to a variety of stimuli  <cit> .
among the tcss identified in the genomes of s. epidermidis, functions of lytsr are unknown, though in s. aureus lytsr has been demonstrated to play a role in bacterial autolysis and biofilm formation.
lytsr two-component regulatory system was firstly identified from the s. aureus genome.
the lyts integration mutant of s. aureus strain nctc 8325- <dig> exhibited a marked propensity to form aggregates in liquid culture and an increased rate of penicillin-and triton x-100-induced lysis.
in combination with subsequent zymographic analysis, it was suggested that lytsr is involved in either regulation of murein hydrolases gene expression or modulation of murein hydrolase activity  <cit> .
recently, shrama et al.
reported that a lyts knockout mutant of s. aureus strain uams- <dig> produced more adherent biofilm  <cit> .
in search of genes regulated by lytsr in s. aureus, two additional open reading frames immediately downstream from lyts and lytr were identified and designated gene lrga and lrgb, whose transcription was positively regulated by lytsr and the global regulators agr and sara.
it was proposed that lrga, and possibly lrgb, functions in a similar way to an antiholin, i.e., blocking murein hydrolases access to the substrate peptidoglycan  <cit> .
bayles et al.
put forward the possibility that lrgab exploits a molecular strategy, which is functionally analogous to that mediated by the eukaryotic bcl- <dig> family of apoptosis regulatory proteins, to control bacterial programmed cell death  <cit> .
recent study suggested that lytsr regulatory system sense a collapse in membrane potential and then induce the transcription of the lrgab operon  <cit> .
several tcss of s. aureus, such as agr and arlrs, have been proven to affect biofilm formation, whereas little has been known in the case of s. epidermidis.
in s. aureus and s. epidermidis, an agr mutant forms a significantly thicker biofilm.
however, the agr regulons of the two species comprise different genes.
autolysin e  which has been documented to mediate initial attachment of s. epidermidis to a polymer surface, overexpresses in an agr mutant, whereas the homologus atl protein in s. aureus is not under agr control  <cit> .
previous studies have shown that arls mutation in s. aureus enhanced biofilm formation on a polystyrene surface in a complex tsb medium  <cit> .
however, an arls knockout mutant of s. epidermidis generated by our laboratory displayed significantly reduced ability of biofilm formation  <cit> , which suggest s. aureus and s. epidermidis adopt different strategies to regulate biofilm formation even though the genome of s. epidermidis is highly homologous to that of s. aureus  <cit> .
therefore, to investigate the role of lytsr in bacterial autolysis and biofilm development in s. epidermidis, 1457Δlytsrstrain was constructed.
the transcriptional profile of 1457Δlytsr was subsequently analyzed by dna microarray and related functions were examined.
RESULTS
construction of s. epidermidis 1457Δlytsr and the complementation strain
because lytsr has been identified as a regulator of autolysis in s. aureus, we hypothesized that lytsr control the rate of autolysis in s. epidermidis, and may be related with biofilm formation.
to test the possibility, lytsr knock-out strategy was applied.
s. epidermidis  <dig> was used in the present study.
we firstly analyzed lytsr operon in s. epidermidis stains rp62a, atcc <dig>  and  <dig>  the lytsr operon was amplified from s. epidermidis  <dig> by pcr with the primers designed according to the s. epidermidis rp62a genome sequence, and shares more than 99% nucleotide identity with that in s. epidermidis strains rp62a and atcc <dig>  blast searches indicated that the lytsr operon is extensively distributed in gram-positive bacteria.
immediately downstream of lytr locates the lrgab operon predicted to encode two potential membrane associated proteins that are similar to bacteriophage holin proteins , as found in s. aureus  <cit> .
the lytsr knockout mutant of s. epidermidis  <dig> was generated by allelic replacement, wherein the ermb gene replaced the predicted histidine kinase domain of lyts and lytr gene .
the lytsr knockout mutant was then verified by direct pcr sequencing  and biochemical tests .
to rule out an influence of second site mutations on the following findings, the complementation plasmid pns-lytsr was constructed and then electroporated into the mutant, whereas introducing the empty vector pns as a negative control.
deletion of lytsr did not result in a significant growth defect, indicating that lytsr is not essential for bacterial cell growth .
the morphology of 1457Δlytsr in stationary phase was observed with transmission electron microscope.
it revealed that the cell surface was rough and diffused, suggesting alterations in its cell wall surface components .
except for diffused cell surface, the Δatle strain had a remarkably thickened cell wall .
modulation of lytsr on murein hydrolase activity
it has been reported that in s. aureus lytsr mutation increased susceptibility to triton x- <dig> induced autolysis, therefore, we investigated effect of lytsr knockout on autolysis in s. epidermidis.
triton x- <dig> induced autolysis of bacterial cells was carried out, the atle knockout mutant as a negative control.
no difference was found between 1457Δlytsr and its parent strain in the triton x- <dig> induced autolysis, inconsistent with that observed in s. aureus  <cit> , while the negative control atle knockout mutant was resistant to autolysis .
given that the lyts mutation in s. aureus has pleiotropic effects on different murein hydrolase activity, zymographic analysis using sds-page incorporated with 2% w/v m. luteus  or s. epidermidis  cells was performed to analyze the activities of extracelluar and cell wall-associated murein hydrolases isolated from bacterial stationary-phase cultures.
no significant difference was observed in the zymographic pattern of murein hydrolases between 1457Δlytsr and the parent strain, regardless of m. luteus or s. epidermidis being taken as the main indicator.
quantitative murein hydrolase assay was further carried out by adding  <dig> μg of extracellular protein extract to a suspension of heat-killed m. luteus or s. epidermidis in tris-hcl buffer, and monitoring the reduction in the suspension turbidity .
however, cell wall hydrolysis performed with extracellular murein hydrolases from 1457Δlytsrwas undergoing more slowly than that from the parent strain.
after  <dig> hours' incubation, a decrease of 69% or 44% in turbidity  was observed in the suspension of m. luteus  or s. epidermidis  added with extracellular murein hydrolases from 1457Δlytsr, contrasted to a reduction of 84% or 54% with extracellular murein hydrolases from the parent strain, indicating that disruption of lytsr resulted in decreased activities of extracellular murein hydrolases  which probably could not be detected by zymographic analysis.
expression of lytsr in trans restored extracellular murein hydrolase activity to nearly wild-type levels .
impact of lytsr knockout on s. epidermidis biofilm formation
as biofilm formation is the major determinant of s.epidermidis pathogenicity, the impact of lytsr deletion on biofilm formation was further investigated.
semi-quantitative assay of s.epidermidis biofilm formation in polystyrene microtitre plates was performed and s.epidermidis atcc <dig> was used as a biofilm negative control.
it was observed that 1457Δlytsr produced slightly more biofilm than the wild-type counterpart .
when lytsr was complemented in the mutant, biofilm formation was reduced to the same levels as that observed in the parent strain .
we further examined cell viability inside biofilm of 1457Δlytsr and the wild-type strain by using a fluorescence-based live/dead staining method.
with an appropriate mixture  of the syto  <dig>  and pi , bacteria with intact cell membranes were stained fluorescent green, whereas bacteria with damaged membranes were stained fluorescent red.
significantly decreased level of red fluorescence was observed inside biofilm of 1457Δlytsr, comparing with that inside biofilm of the wild-type strain, as shown in figure  <dig>  complementation of 1457Δlytsr with plasmid pns-lytsr restored the level of red fluorescence to that observed inside biofilm of the wild-type strain .
a quantitative method based on measuring the red/green fluorescence ratio was carried out to determine the relative cell viability inside biofilm.
the percentage of dead cells inside 24-hour-old biofilms of 1457Δlytsr and the wild-type strain were 6% and 15% respectively, as shown in figure  <dig>  inside the biofilm of lytsr complementation strain, the percentage of dead cells was restored nearly to the wild-type level.
transcriptional profiling of 1457Δlytsr strain
to investigate the regulatory role of lytsr, we used custom-made s. epidermidis genechips to perform a transcriptional profile analysis of the wild type and 1457Δlytsr strains.
two criteria including 2-fold or greater change in expression level and p <  <dig>  were employed to select the genes with significantly different expression.
it was found that expression of  <dig> genes was affected by lytsr mutation, in which  <dig> were upregulated and  <dig> were downregulated.
transcription of lrgab decreased drastically in 1457Δlytsr, indicating that the operon was activated by lytsr in s.epidermidis, consistent with the finding for s. aureus.
further analysis of the microarray data showed that genes upregulated in the 1457Δlytsr strain included these involved in purine biosynthesis , amino acid biosynthesis  and membrane transport .
whereas, genes downregulated contained these involved in pyruvate metabolism , anaerobic growth  .
in addition, genes responsible for encoding ribosomal proteins which make up the ribosomal subunits in conjunction with rrna were found to be downregulated in 1457Δlytsr , consistent with that reported in transcriptional profiling studies of s. aureus by sharma et al.
<cit> .
transcription of lrgab decreased drastically in 1457Δlytsr, indicating that the operon was activated by lytsr in s.epidermidis, consistent with the finding for s. aureus.
we also noticed that expression of an arac family transcriptional regulator homologue was remarkably higher in the mutant .
the microarray experiments were repeated by prof. jacques schrenzel .
transcription of genes required for amino acid biosynthesis, carbon metabolism and membrane transport was also found to be altered in the mutant.
moreover, differential expression of general stress protein, alkaline shock protein  <dig> and cold shock protein was observed in the latter microarray data.
taken together, it suggested that lytsr may be involved in sensing and responding to changes in the metabolic state of the bacteria.
* the entire list of differentially expressed genes can be found on the national center for biotechnology information gene expression omnibus  in 1457Δlytsr were confirmed by real-time rt-pcr with gyrb, a housekeeping gene, as the internal control, as shown in table  <dig>
* data are means ± sd of  <dig> independent experiments.
***p <  <dig> ; **p <  <dig> ; Δytsr <dig> vs. wt.
pyruvate utilization of  <dig> and 1457Δlytsr
ability of 1457Δlytsrto utilize pyruvate was found to be impaired by using the vitek gpi card system.
meanwhile, expression of genes involved in pyruvate metabolism such as mqo- <dig>  mqo- <dig> and its neighboring unknown gene serp <dig> were remarkably reduced.
for examining the ability to utilize pyruvate, strains  <dig> and 1457Δlytsrwere cultured in pyruvate fermentation broth and bacterial growth was monitored.
the 1457Δlytsr displayed a significantly growth defect in pyruvate fermentation broth, whereas introducing plasmid pns-lytsr into the mutant restored the phenotype, as shown in figure  <dig>
discussion
the capacity of staphylococci to produce a biofilm is determined by environmental factors, such as glucose, osmolarity, ethanol, temperature and anaerobiosis etc, which suggests that there is a mechanism that senses and responds to extracellular signals  <cit> .
two-component regulatory systems, composed of histidine kinases and their cognate response regulators, are the predominant means by which bacteria adapt to changes in their environment  <cit> .
previous studies have shown yycg/yycf two-component system is essential for cell viability in b. subtilis and s. aureus and positively controls biofilm formation  <cit> .
another two tcss of s. aureus, agr and arlrs, have also been proven to regulate biofilm formation  <cit> .
seventeen pairs of tcss have been determined in the genome of s. epidermidis atcc <dig> , while  <dig> pairs in atcc <dig>  <cit> .
we identified one pair of tcs encoding lyts and lytr homologs described in s. aureus  <cit> .
the lytsr two-component system in s. aureus has been viewed as an important regulator of bacterial autolysis  <cit> .
in the present study, the function of the s. epidermidis lytsr opreon was firstly investigated.
the lytsr knockout mutation did not alter the susceptibility of strain  <dig> to triton x-100-induced lysis, which is different from the finding for s. aureus strain nctc 8325- <dig> reported by brunskill et al.
<cit> .
recently, they found that in the strain uams- <dig>  lyts knock-out did not result in spontaneous and triton x-100-induced lysis increasing  <cit> .
the variation in susceptibility to triton x-100-induced lysis between different staphylococcus strains could be explained partly by the fact that they represent different genetic background.
since that lyts mutation in s. aureus has pleiotropic effects on different murein hydrolase activity  <cit> , we hypothesized that in s. epidermidis, lytsr regulates murein hydrolase activity in a similar manner.
zymographic analysis revealed no significant differences between 1457Δlytsr and the parent strain in the activities or expression of murein hydrolase isolated from both extracellular and cell wall fraction.
however, quantification of the extracellular murein hydrolase activity produced by these strains demonstrated that 1457Δlytsr produced diminished overall activity compared to that of the parental strain.
as expected, microarray analysis revealed that lrgab opreon was downregulated in 1457Δlytsr.
in s. aureus, lrgab has a negative regulatory effect on extracellular murein hydrolase activity and disruption of lrgab led to a significant increase in the activity  <cit> .
cidab operon, which encodes the holin-like counterpart of the lrgab operon, and alssd operon, which encodes proteins involved in acetoin production, were then identified.
mutation of either cidab or alssd operon in the s. aureus strain uams- <dig> caused a dramatic decrease in extracellular murein hydrolase activity  <cit> .
we, therefore, speculate that in s. epidermidis some other lytsr regulated proteins similar to cidab and/or alssd, may exist and overcome negative effect imposed by lrgab on extracellular murein hydrolase activity, which warrants further investigation.
the role of cell death and lysis in bacterial adaptive responses to circumstances has been well elucidated in a number of bacteria, such as s. aureus and p. aeruginosa.
webb et al.
proposed that in p. aeruginosa cell death benefited a subpopulation of surviving cells and therefore facilitated subsequent biofilm differentiation and dispersal  <cit> .
moreover, genomic dna released following bacterial lysis constitutes the skeleton of biofilm.
since lytsr positively regulates the activity of extracellular murein hydrolases, it may affect cell viability and function in biofilm formation.
by using the clsm, significant decrease in red fluorescence was observed inside biofilm of 1457Δlytsr, which indicated reduced loss of cell viability.
quantitative analysis showed that the percentage of dead cells inside biofilm of the wild type strain was approximately two times higher than that in the mutant.
the results are consistent with the observation that 1457Δlytsr displayed a reduction in activity of extracellular murein hydrolases.
disruption of either cida or alssd genes on the s. aureus chromosome resulted in significantly decreased extracellular murein hydrolase activity compared with that of the parental strain, uams- <dig>  both the cida and the alssd mutant displayed reduced cell death in stationary phase and completely abrogated cell lysis relative to uams- <dig>  <cit> .
along these lines, the present study confirmed a connection between extracellular murein hydrolase activity and bacterial cell death.
furthermore, expression of cidc gene encoding pyruvate oxidase was found to be downregulated  in 1457Δlytsr through the microarray analysis.
deletion of cidc in s. aureus or s. pneumoniae caused reduced cell death and lysis in stationary phase <cit> .
based on these data, it was suggested lytsr may play an important role in bacterial cell death and lysis inside biofilm.
in this study, 1457Δlytsrwas found to have growth defect in pyruvate fermentation broth and introducing plasmid encoding lytsr  into the mutant completely restored the phenotype.
based on the fact that the wild-type strain and the mutant grow equally well in tsb containing  <dig> % glucose.
as we know, glucose is catabolized by glycolysis to pyruvate.
if 1457Δlytsris impaired in its ability to metabolize pyruvate, then this would be reflected in the growth curve in tsb medium.
the data actually indicated that 1457Δlytsris impaired in the transport of pyruvate and probably amino acids.
previous studies regarding bacterial cells taking up carboxylic acid from the surrounding medium have shown that pyruvate is actively transported across the bacterial membrane and that proton motive force  plays an important role in the process  <cit> .
in addition, transcription of genes involved in pyruvate metabolism such as mqo- <dig>  mqo- <dig> and its neighbouring unknown gene serp <dig> were significantly downregulated in 1457Δlytsr.
these data along with the findings that in s. aureus lytsr responds to a collapse in Δψ by inducing the transcription of the lrgab operon led us to hypothesize that lytsr accelerates pyruvate transport by sensing a reduction in pmf.
compared to the parent stain, 1457Δlytsrexhibited decreased expression of ribosomal genes and increased expression of amino acid biosynthetic genes, amino acyl-trna synthase genes, and amino acid transporters genes, which implies that lytsr mutation may induce a stringent response.
additionally, transcriptional profiling studies performed in switzerland revealed that expression level of genes involved in stress response and cold shock was altered in the mutant.
when bacteria encounter sudden unfavorable environment, protein synthesis will be inhibited, causing the induction or repression of many metabolic pathways according to physiological needs, and the induction of stationary-phase survival genes.
this is called "the stringent response".
bacterial alarmone ppgpp functions as a global regulator responsible for the stringent control.
two homologous ppgpp synthetases, rela and spot, have been identified and characterized in escherichia coli  <cit> .
lemos et al.
have reported that the rela mutation impaired the capacity of streptococcus mutans to form biofilm <cit> .
no changes in transcription of the rela/spot homolog were found in 1457Δlytsr.
however, serp <dig> encoding an arac family transcriptional regulator was found to be upregulated significantly in the mutant.
transcriptional regulators of the arac family are widespread among bacteria and have three main regulatory functions in common: carbon metabolism, stress response, and pathogenesis <cit> .
among the microarray data, several genes predicted to be involved in anaerobic metabolism were of particular interest.
the arc operon encodes the enzymes of the arginine deiminase  pathway, which catalyzes the conversion of arginine into ornithine, ammonia, and co <dig>  with the concomitant production of  <dig> mol of atp per mol of arginine consumed.
in the absence of oxygen, the adi pathway enables s. aureus to grow in the medium containing arginine  <cit> .
recent studies demonstrated that the arc operon identified in the genome of s epidermidis strain atcc <dig> but not in rp62a is located on a novel genomic island termed arginine catabolic mobile element .
except for the acme-encoded arc operon, all s. epidermidis carry a native arc operon on the core chromosome.
diep et al.
supposed that acme-encoded gene products might confer survival advantage of s. aureus strain usa <dig> and other acme-bearing staphylococci within the host, resulting in the widespread dissemination of bacterial progeny  <cit> .
in the present study, arginine deiminase activity was performed as previously described  <cit>  and 1457Δlytsr exhibited a reduced enzyme activity .
in the present study, 1457Δlytsr produced slightly more biofilm than its parent strain.
however, no genes that are involved in biofilm formation directly, such as ica operon encoding enzymes responsible for pia synthesis, were identified in the transcriptional profile.
it was observed that ica transcription level and pia production were similar between 1457Δlytsr and its parent strain.
both tricarboxylic acid cycle stress and anaerobic condition have been proven to induce pia production and promotion of biofilm, suggesting that changes in the metabolic status can be sensed and regulate biofilm formation  <cit> .
moreover, the stringent response has also been demonstrated to affect biofilm formation <cit> .
it suggests that lytsr mutation may indirectly enhance biofilm formation by altering the metabolic status of s. epidermidis.
CONCLUSIONS
the present study suggests that in s. epidermidis the lytsr two-component regulatory system play an important role in controlling extracellular murein hydrolase activity and bacterial cell death but has limited effect on autolysis.
the lytsr mutation invokes a stringent type transcriptional profile, moreover, enhances biofilm formation, which suggests lytsr may function to indirectly regulate biofilm formation by altering the metabolic status of the bacteria, particularly under conditions in which supply of nutrient and oxygen is limited, such as the conditions in biofilm.

 staphylococcus epidermidis is an opportunistic pathogen which normally inhabits human skin and mucous membranes, primarily infecting immunocompromised individuals or those with implanted biomaterials. the pathogenicity of s. epidermidis is mostly due to its ability to form a thick, multilayered biofilm on polymeric surfaces. treatment of s. epidermidis infection has become a troublesome problem as biofilm-associated bacteria exhibit enhanced resistance to antibiotics and to components of the innate host defences. among the staphylococci, the other major human pathogen is staphylococcus aureus, which causes infections ranging from cutaneous infections and food poisoning to life-threatening septicaemia. aside from biofilm, s. aureus produce a large array of exotoxins and exoezymes. two-component regulatory systems play a pivotal role in bacterial adaptation, survival, and virulence by sensing changes in the external environment and modulating gene expression in response to a variety of stimuli. among the tcss identified in the genomes of s. epidermidis, functions of lytsr are unknown, though in s. aureus lytsr has been demonstrated to play a role in bacterial autolysis and biofilm formation. lytsr two-component regulatory system was firstly identified from the s. aureus genome. the lyts integration mutant of s. aureus strain nctc 8325- exhibited a marked propensity to form aggregates in liquid culture and an increased rate of penicillin-and triton x-100-induced lysis. in combination with subsequent zymographic analysis, it was suggested that lytsr is involved in either regulation of murein hydrolases gene expression or modulation of murein hydrolase activity. recently, shrama et al. reported that a lyts knockout mutant of s. aureus strain uams- produced more adherent biofilm. in search of genes regulated by lytsr in s. aureus, two additional open reading frames immediately downstream from lyts and lytr were identified and designated gene lrga and lrgb, whose transcription was positively regulated by lytsr and the global regulators agr and sara. it was proposed that lrga, and possibly lrgb, functions in a similar way to an antiholin, i.e., blocking murein hydrolases access to the substrate peptidoglycan. bayles et al. put forward the possibility that lrgab exploits a molecular strategy, which is functionally analogous to that mediated by the eukaryotic bcl- family of apoptosis regulatory proteins, to control bacterial programmed cell death. recent study suggested that lytsr regulatory system sense a collapse in membrane potential and then induce the transcription of the lrgab operon. several tcss of s. aureus, such as agr and arlrs, have been proven to affect biofilm formation, whereas little has been known in the case of s. epidermidis. in s. aureus and s. epidermidis, an agr mutant forms a significantly thicker biofilm. however, the agr regulons of the two species comprise different genes. autolysin e which has been documented to mediate initial attachment of s. epidermidis to a polymer surface, overexpresses in an agr mutant, whereas the homologus atl protein in s. aureus is not under agr control. previous studies have shown that arls mutation in s. aureus enhanced biofilm formation on a polystyrene surface in a complex tsb medium. however, an arls knockout mutant of s. epidermidis generated by our laboratory displayed significantly reduced ability of biofilm formation, which suggest s. aureus and s. epidermidis adopt different strategies to regulate biofilm formation even though the genome of s. epidermidis is highly homologous to that of s. aureus. therefore, to investigate the role of lytsr in bacterial autolysis and biofilm development in s. epidermidis, 1457Δlytsrstrain was constructed. the transcriptional profile of 1457Δlytsr was subsequently analyzed by dna microarray and related functions were examined.  construction of s. epidermidis 1457Δlytsr and the complementation strain because lytsr has been identified as a regulator of autolysis in s. aureus, we hypothesized that lytsr control the rate of autolysis in s. epidermidis, and may be related with biofilm formation. to test the possibility, lytsr knock-out strategy was applied. s. epidermidis was used in the present study. we firstly analyzed lytsr operon in s. epidermidis stains rp62a, atcc and the lytsr operon was amplified from s. epidermidis by pcr with the primers designed according to the s. epidermidis rp62a genome sequence, and shares more than 99% nucleotide identity with that in s. epidermidis strains rp62a and atcc blast searches indicated that the lytsr operon is extensively distributed in gram-positive bacteria. immediately downstream of lytr locates the lrgab operon predicted to encode two potential membrane associated proteins that are similar to bacteriophage holin proteins, as found in s. aureus. the lytsr knockout mutant of s. epidermidis was generated by allelic replacement, wherein the ermb gene replaced the predicted histidine kinase domain of lyts and lytr gene. the lytsr knockout mutant was then verified by direct pcr sequencing and biochemical tests. to rule out an influence of second site mutations on the following findings, the complementation plasmid pns-lytsr was constructed and then electroporated into the mutant, whereas introducing the empty vector pns as a negative control. deletion of lytsr did not result in a significant growth defect, indicating that lytsr is not essential for bacterial cell growth. the morphology of 1457Δlytsr in stationary phase was observed with transmission electron microscope. it revealed that the cell surface was rough and diffused, suggesting alterations in its cell wall surface components. except for diffused cell surface, the Δatle strain had a remarkably thickened cell wall. modulation of lytsr on murein hydrolase activity it has been reported that in s. aureus lytsr mutation increased susceptibility to triton x- induced autolysis, therefore, we investigated effect of lytsr knockout on autolysis in s. epidermidis. triton x- induced autolysis of bacterial cells was carried out, the atle knockout mutant as a negative control. no difference was found between 1457Δlytsr and its parent strain in the triton x- induced autolysis, inconsistent with that observed in s. aureus, while the negative control atle knockout mutant was resistant to autolysis. given that the lyts mutation in s. aureus has pleiotropic effects on different murein hydrolase activity, zymographic analysis using sds-page incorporated with 2% w/v m. luteus or s. epidermidis cells was performed to analyze the activities of extracelluar and cell wall-associated murein hydrolases isolated from bacterial stationary-phase cultures. no significant difference was observed in the zymographic pattern of murein hydrolases between 1457Δlytsr and the parent strain, regardless of m. luteus or s. epidermidis being taken as the main indicator. quantitative murein hydrolase assay was further carried out by adding μg of extracellular protein extract to a suspension of heat-killed m. luteus or s. epidermidis in tris-hcl buffer, and monitoring the reduction in the suspension turbidity. however, cell wall hydrolysis performed with extracellular murein hydrolases from 1457Δlytsrwas undergoing more slowly than that from the parent strain. after hours' incubation, a decrease of 69% or 44% in turbidity was observed in the suspension of m. luteus or s. epidermidis added with extracellular murein hydrolases from 1457Δlytsr, contrasted to a reduction of 84% or 54% with extracellular murein hydrolases from the parent strain, indicating that disruption of lytsr resulted in decreased activities of extracellular murein hydrolases which probably could not be detected by zymographic analysis. expression of lytsr in trans restored extracellular murein hydrolase activity to nearly wild-type levels. impact of lytsr knockout on s. epidermidis biofilm formation as biofilm formation is the major determinant of s.epidermidis pathogenicity, the impact of lytsr deletion on biofilm formation was further investigated. semi-quantitative assay of s.epidermidis biofilm formation in polystyrene microtitre plates was performed and s.epidermidis atcc was used as a biofilm negative control. it was observed that 1457Δlytsr produced slightly more biofilm than the wild-type counterpart. when lytsr was complemented in the mutant, biofilm formation was reduced to the same levels as that observed in the parent strain. we further examined cell viability inside biofilm of 1457Δlytsr and the wild-type strain by using a fluorescence-based live/dead staining method. with an appropriate mixture of the syto and pi, bacteria with intact cell membranes were stained fluorescent green, whereas bacteria with damaged membranes were stained fluorescent red. significantly decreased level of red fluorescence was observed inside biofilm of 1457Δlytsr, comparing with that inside biofilm of the wild-type strain, as shown in figure complementation of 1457Δlytsr with plasmid pns-lytsr restored the level of red fluorescence to that observed inside biofilm of the wild-type strain. a quantitative method based on measuring the red/green fluorescence ratio was carried out to determine the relative cell viability inside biofilm. the percentage of dead cells inside 24-hour-old biofilms of 1457Δlytsr and the wild-type strain were 6% and 15% respectively, as shown in figure inside the biofilm of lytsr complementation strain, the percentage of dead cells was restored nearly to the wild-type level. transcriptional profiling of 1457Δlytsr strain to investigate the regulatory role of lytsr, we used custom-made s. epidermidis genechips to perform a transcriptional profile analysis of the wild type and 1457Δlytsr strains. two criteria including 2-fold or greater change in expression level and p < were employed to select the genes with significantly different expression. it was found that expression of genes was affected by lytsr mutation, in which were upregulated and were downregulated. transcription of lrgab decreased drastically in 1457Δlytsr, indicating that the operon was activated by lytsr in s.epidermidis, consistent with the finding for s. aureus. further analysis of the microarray data showed that genes upregulated in the 1457Δlytsr strain included these involved in purine biosynthesis, amino acid biosynthesis and membrane transport. whereas, genes downregulated contained these involved in pyruvate metabolism, anaerobic growth. in addition, genes responsible for encoding ribosomal proteins which make up the ribosomal subunits in conjunction with rrna were found to be downregulated in 1457Δlytsr, consistent with that reported in transcriptional profiling studies of s. aureus by sharma et al.. transcription of lrgab decreased drastically in 1457Δlytsr, indicating that the operon was activated by lytsr in s.epidermidis, consistent with the finding for s. aureus. we also noticed that expression of an arac family transcriptional regulator homologue was remarkably higher in the mutant. the microarray experiments were repeated by prof. jacques schrenzel. transcription of genes required for amino acid biosynthesis, carbon metabolism and membrane transport was also found to be altered in the mutant. moreover, differential expression of general stress protein, alkaline shock protein and cold shock protein was observed in the latter microarray data. taken together, it suggested that lytsr may be involved in sensing and responding to changes in the metabolic state of the bacteria. * the entire list of differentially expressed genes can be found on the national center for biotechnology information gene expression omnibus in 1457Δlytsr were confirmed by real-time rt-pcr with gyrb, a housekeeping gene, as the internal control, as shown in table * data are means ± sd of independent experiments. ***p <; **p <; Δytsr vs. wt. pyruvate utilization of and 1457Δlytsr ability of 1457Δlytsrto utilize pyruvate was found to be impaired by using the vitek gpi card system. meanwhile, expression of genes involved in pyruvate metabolism such as mqo- mqo- and its neighboring unknown gene serp were remarkably reduced. for examining the ability to utilize pyruvate, strains and 1457Δlytsrwere cultured in pyruvate fermentation broth and bacterial growth was monitored. the 1457Δlytsr displayed a significantly growth defect in pyruvate fermentation broth, whereas introducing plasmid pns-lytsr into the mutant restored the phenotype, as shown in figure discussion the capacity of staphylococci to produce a biofilm is determined by environmental factors, such as glucose, osmolarity, ethanol, temperature and anaerobiosis etc, which suggests that there is a mechanism that senses and responds to extracellular signals. two-component regulatory systems, composed of histidine kinases and their cognate response regulators, are the predominant means by which bacteria adapt to changes in their environment. previous studies have shown yycg/yycf two-component system is essential for cell viability in b. subtilis and s. aureus and positively controls biofilm formation. another two tcss of s. aureus, agr and arlrs, have also been proven to regulate biofilm formation. seventeen pairs of tcss have been determined in the genome of s. epidermidis atcc, while pairs in atcc. we identified one pair of tcs encoding lyts and lytr homologs described in s. aureus. the lytsr two-component system in s. aureus has been viewed as an important regulator of bacterial autolysis. in the present study, the function of the s. epidermidis lytsr opreon was firstly investigated. the lytsr knockout mutation did not alter the susceptibility of strain to triton x-100-induced lysis, which is different from the finding for s. aureus strain nctc 8325- reported by brunskill et al.. recently, they found that in the strain uams- lyts knock-out did not result in spontaneous and triton x-100-induced lysis increasing. the variation in susceptibility to triton x-100-induced lysis between different staphylococcus strains could be explained partly by the fact that they represent different genetic background. since that lyts mutation in s. aureus has pleiotropic effects on different murein hydrolase activity, we hypothesized that in s. epidermidis, lytsr regulates murein hydrolase activity in a similar manner. zymographic analysis revealed no significant differences between 1457Δlytsr and the parent strain in the activities or expression of murein hydrolase isolated from both extracellular and cell wall fraction. however, quantification of the extracellular murein hydrolase activity produced by these strains demonstrated that 1457Δlytsr produced diminished overall activity compared to that of the parental strain. as expected, microarray analysis revealed that lrgab opreon was downregulated in 1457Δlytsr. in s. aureus, lrgab has a negative regulatory effect on extracellular murein hydrolase activity and disruption of lrgab led to a significant increase in the activity. cidab operon, which encodes the holin-like counterpart of the lrgab operon, and alssd operon, which encodes proteins involved in acetoin production, were then identified. mutation of either cidab or alssd operon in the s. aureus strain uams- caused a dramatic decrease in extracellular murein hydrolase activity. we, therefore, speculate that in s. epidermidis some other lytsr regulated proteins similar to cidab and/or alssd, may exist and overcome negative effect imposed by lrgab on extracellular murein hydrolase activity, which warrants further investigation. the role of cell death and lysis in bacterial adaptive responses to circumstances has been well elucidated in a number of bacteria, such as s. aureus and p. aeruginosa. webb et al. proposed that in p. aeruginosa cell death benefited a subpopulation of surviving cells and therefore facilitated subsequent biofilm differentiation and dispersal. moreover, genomic dna released following bacterial lysis constitutes the skeleton of biofilm. since lytsr positively regulates the activity of extracellular murein hydrolases, it may affect cell viability and function in biofilm formation. by using the clsm, significant decrease in red fluorescence was observed inside biofilm of 1457Δlytsr, which indicated reduced loss of cell viability. quantitative analysis showed that the percentage of dead cells inside biofilm of the wild type strain was approximately two times higher than that in the mutant. the results are consistent with the observation that 1457Δlytsr displayed a reduction in activity of extracellular murein hydrolases. disruption of either cida or alssd genes on the s. aureus chromosome resulted in significantly decreased extracellular murein hydrolase activity compared with that of the parental strain, uams- both the cida and the alssd mutant displayed reduced cell death in stationary phase and completely abrogated cell lysis relative to uams-. along these lines, the present study confirmed a connection between extracellular murein hydrolase activity and bacterial cell death. furthermore, expression of cidc gene encoding pyruvate oxidase was found to be downregulated in 1457Δlytsr through the microarray analysis. deletion of cidc in s. aureus or s. pneumoniae caused reduced cell death and lysis in stationary phase. based on these data, it was suggested lytsr may play an important role in bacterial cell death and lysis inside biofilm. in this study, 1457Δlytsrwas found to have growth defect in pyruvate fermentation broth and introducing plasmid encoding lytsr into the mutant completely restored the phenotype. based on the fact that the wild-type strain and the mutant grow equally well in tsb containing % glucose. as we know, glucose is catabolized by glycolysis to pyruvate. if 1457Δlytsris impaired in its ability to metabolize pyruvate, then this would be reflected in the growth curve in tsb medium. the data actually indicated that 1457Δlytsris impaired in the transport of pyruvate and probably amino acids. previous studies regarding bacterial cells taking up carboxylic acid from the surrounding medium have shown that pyruvate is actively transported across the bacterial membrane and that proton motive force plays an important role in the process. in addition, transcription of genes involved in pyruvate metabolism such as mqo- mqo- and its neighbouring unknown gene serp were significantly downregulated in 1457Δlytsr. these data along with the findings that in s. aureus lytsr responds to a collapse in Δψ by inducing the transcription of the lrgab operon led us to hypothesize that lytsr accelerates pyruvate transport by sensing a reduction in pmf. compared to the parent stain, 1457Δlytsrexhibited decreased expression of ribosomal genes and increased expression of amino acid biosynthetic genes, amino acyl-trna synthase genes, and amino acid transporters genes, which implies that lytsr mutation may induce a stringent response. additionally, transcriptional profiling studies performed in switzerland revealed that expression level of genes involved in stress response and cold shock was altered in the mutant. when bacteria encounter sudden unfavorable environment, protein synthesis will be inhibited, causing the induction or repression of many metabolic pathways according to physiological needs, and the induction of stationary-phase survival genes. this is called "the stringent response". bacterial alarmone ppgpp functions as a global regulator responsible for the stringent control. two homologous ppgpp synthetases, rela and spot, have been identified and characterized in escherichia coli. lemos et al. have reported that the rela mutation impaired the capacity of streptococcus mutans to form biofilm. no changes in transcription of the rela/spot homolog were found in 1457Δlytsr. however, serp encoding an arac family transcriptional regulator was found to be upregulated significantly in the mutant. transcriptional regulators of the arac family are widespread among bacteria and have three main regulatory functions in common: carbon metabolism, stress response, and pathogenesis. among the microarray data, several genes predicted to be involved in anaerobic metabolism were of particular interest. the arc operon encodes the enzymes of the arginine deiminase pathway, which catalyzes the conversion of arginine into ornithine, ammonia, and co with the concomitant production of mol of atp per mol of arginine consumed. in the absence of oxygen, the adi pathway enables s. aureus to grow in the medium containing arginine. recent studies demonstrated that the arc operon identified in the genome of s epidermidis strain atcc but not in rp62a is located on a novel genomic island termed arginine catabolic mobile element. except for the acme-encoded arc operon, all s. epidermidis carry a native arc operon on the core chromosome. diep et al. supposed that acme-encoded gene products might confer survival advantage of s. aureus strain usa and other acme-bearing staphylococci within the host, resulting in the widespread dissemination of bacterial progeny. in the present study, arginine deiminase activity was performed as previously described and 1457Δlytsr exhibited a reduced enzyme activity. in the present study, 1457Δlytsr produced slightly more biofilm than its parent strain. however, no genes that are involved in biofilm formation directly, such as ica operon encoding enzymes responsible for pia synthesis, were identified in the transcriptional profile. it was observed that ica transcription level and pia production were similar between 1457Δlytsr and its parent strain. both tricarboxylic acid cycle stress and anaerobic condition have been proven to induce pia production and promotion of biofilm, suggesting that changes in the metabolic status can be sensed and regulate biofilm formation. moreover, the stringent response has also been demonstrated to affect biofilm formation. it suggests that lytsr mutation may indirectly enhance biofilm formation by altering the metabolic status of s. epidermidis.  the present study suggests that in s. epidermidis the lytsr two-component regulatory system play an important role in controlling extracellular murein hydrolase activity and bacterial cell death but has limited effect on autolysis. the lytsr mutation invokes a stringent type transcriptional profile, moreover, enhances biofilm formation, which suggests lytsr may function to indirectly regulate biofilm formation by altering the metabolic status of the bacteria, particularly under conditions in which supply of nutrient and oxygen is limited, such as the conditions in biofilm. 
impaired ability to utilize pyruvate and reduced activity of arginine deiminase was observed in 1457Δlytsr, which is consistent with the microarray data. staphylococcus epidermidis has emerged as one of the most important nosocomial pathogens, mainly because of its ability to colonize implanted biomaterials by forming a biofilm. extensive studies are focused on the molecular mechanisms involved in biofilm formation. the lytsr two-component regulatory system regulates autolysis and biofilm formation in staphylococcus aureus. in addition, lytsr may indirectly enhance biofilm formation by altering the metabolic status of the bacteria. the preliminary results suggest that in s. epidermidis lytsr two-component system regulates extracellular murein hydrolase activity, bacterial cell death and pyruvate utilization. microarray analysis showed that lytsr mutation affected the transcription of genes. compared to the wild-type counterpart, 1457Δlytsr produced slightly more biofilm, with significantly decreased dead cells inside. based on the microarray data, it appears that lytsr inactivation induces a stringent response. quantitative murein hydrolase assay indicated that disruption of lytsr in s. epidermidis resulted in decreased activities of extracellular murein hydrolases, although zymogram showed no apparent differences in murein hydrolase patterns between s. epidermidis strain and its lytsr mutant. however, the role of lytsr played in s. epidermidis remained unknown. in the present study, we demonstrated that lytsr knock-out in s. epidermidis did not alter susceptibility to triton x- induced autolysis. specifically, genes encoding proteins responsible for protein synthesis, energy metabolism were downregulated, while genes involved in amino acid and nucleotide biosynthesis, amino acid transporters were upregulated.
 staphylococcus epidermidis has emerged as one of the most important nosocomial pathogens, mainly because of its ability to colonize implanted biomaterials by forming a biofilm. extensive studies are focused on the molecular mechanisms involved in biofilm formation. the lytsr two-component regulatory system regulates autolysis and biofilm formation in staphylococcus aureus. however, the role of lytsr played in s. epidermidis remained unknown.  in the present study, we demonstrated that lytsr knock-out in s. epidermidis did not alter susceptibility to triton x- induced autolysis. quantitative murein hydrolase assay indicated that disruption of lytsr in s. epidermidis resulted in decreased activities of extracellular murein hydrolases, although zymogram showed no apparent differences in murein hydrolase patterns between s. epidermidis strain and its lytsr mutant. compared to the wild-type counterpart, 1457Δlytsr produced slightly more biofilm, with significantly decreased dead cells inside. microarray analysis showed that lytsr mutation affected the transcription of genes. specifically, genes encoding proteins responsible for protein synthesis, energy metabolism were downregulated, while genes involved in amino acid and nucleotide biosynthesis, amino acid transporters were upregulated. impaired ability to utilize pyruvate and reduced activity of arginine deiminase was observed in 1457Δlytsr, which is consistent with the microarray data.  the preliminary results suggest that in s. epidermidis lytsr two-component system regulates extracellular murein hydrolase activity, bacterial cell death and pyruvate utilization. based on the microarray data, it appears that lytsr inactivation induces a stringent response. in addition, lytsr may indirectly enhance biofilm formation by altering the metabolic status of the bacteria. 