BACKGROUND
the u.s. environmental protection agency  has endeavored to determine the efficacy and the mode of action of antimicrobials.
at epa,  <dig>  antimicrobial products are registered, and hospital-level disinfectants are being tested against pathogens such as s. aureus, which is responsible for many infectious diseases, ranging from benign skin infections to life-threatening endocarditis and toxic shock syndrome  <cit> .
one of the reasons epa has exerted such efforts is that hospital-acquired infections are a serious threat to public health.
therefore, it is important to use appropriate antimicrobial agents with clear understanding of the subsequent effects to prevent infection outbreaks in health care environments  <cit> .
the phenolic compound, ortho-phenylphenol , is an antimicrobial agent and an active ingredient of epa-registered disinfectant with wide human exposure in various agricultural, hospital and veterinary disinfectant products.
opp is employed in a variety of applications, including hard surface disinfection, wood preservation, treatment of citrus fruit, vegetables before packaging to prevent microbial decay and textile production due to its bactericidal and fungicidal activity  <cit> .
there have been several reports related to the exposure of opp on humans.
it has been reported that opp increased the incidence of urinary bladder tumors in f344/ducrj rats when administered in the diet  <cit> .
the results of this study stimulated the initiation of additional testing of opp for both tumor induction and possible reactivity with dna.
opp has been found to have estrogenic or anti-androgenic activity, and binds to the androgen or estrogen receptors  <cit> .
in spite of these effects opp is still used in applications that simultaneously contact both humans and bacteria.
it is therefore important to understand the differential effects on each so that its efficacy can be understood and even optimized.
moreover, a lack of understanding of a cellular response to opp hinders further development of more innovative methods for combating pathogens.
certainly, better elucidation of the molecular events responsible for establishing and maintaining pathogenicity will help to map affected cell functions and serve to delineate the mechanisms involved in the disinfectant activity.
microarrays have been effectively employed to simultaneously and globally examine the complete transcriptional response at the genomic level in pseudomonas aeruginosa and s. aureus upon exposure to antimicrobials  <cit> .
in this study, to our knowledge, for the first time, we show that the global transcription response of s. aureus to opp includes downregulation of genes involved in lysine metabolism, as well as genes involved in amino acid metabolism, by utilizing affymetrix s. aureus genechip arrays.
our findings indicate that:  many cellular protective processes were upregulated,  the transcription of genes involved in primary metabolic pathways was downregulated, and  the transcription of genes encoding lysine and histidine biosynthesis was downregulated.
next we performed real-time pcr analysis on selected genes to validate the array results.
based on this result, it was concluded that this study may help to elucidate the mechanism of action by which opp stops cell wall construction and thereby inhibiting s. aureus growth, and may facilitate the design of more effective antimicrobials.
RESULTS
growth inhibition by opp
to determine the sublethal inhibitory effect of opp on s. aureus, we first exposed the exponentially growing cells to different concentrations of opp dissolved in dmso .
in figure  <dig>  we demonstrate that  <dig>  mm concentration of opp caused a growth inhibition for about  <dig> min.
note that minimum inhibitory concentration  of opp on s. aureus was reportedly  <dig> mg/l   <cit> .
in this study, to better understand how s. aureus initially responds to opp, we chose the rate of cell growth inhibition with  <dig>  mm opp after  <dig> and  <dig> min exposure times compared to control .
transcriptional profiles in response to opp
to investigate early transcriptional changes in response to opp exposure, we isolated total rna after  <dig> min and  <dig> minutes exposure to  <dig>  mm opp and conducted five independent microarray experiments in the absence  and the presence  of  <dig>  mm opp .
to further identify genes with statistically marked changes in expression levels, we applied the following criteria to each of the  <dig> min,  <dig> min, and control-experimental microarray data sets:  a p-value for a t-test should be equal to or less than  <dig> ,  an absolute fold change in transcript level should be equal to or greater than  <dig>  and  a gene should have a presence or marginal call  <cit>  from 50% or more replicates on both the experimental and control replicate sets.
of the  <dig>  genes represented on the s. aureus genechip,  <dig>  genes showed statistical significance based on a 1-way anova.
we found that mrna levels  <dig> genes of s. aureus were significantly altered in response to opp by two fold or more upregulation or downregulation.
the raw data of  <dig>  genes control  and experimental genes after  has been deposited in ncbi's gene expression omnibus  <cit>  and is accessible through geo series accession number gse <dig> .
analysis of gene expression changes in  <dig> min and  <dig> min
to examine how genes with transcript level changes are distributed with regard to their functions, we further classified these  <dig> genes that were either upregulated or downregulated by a fold change of two or more according to the gene classification based on cog functional categories in the genome of national center for biotechnology information   <cit>  .
in figure  <dig>  the differences between the numbers of up and downregulated genes in each functional class after  <dig> and  <dig> minutes exposure to  <dig>  mm of opp are illustrated.
note that figure  <dig> represents a total of  <dig> genes including the group of "function unknown , hypothetical protein  and general function predicted only ".
some interesting findings are as follows:  the genes of amino acid transport and metabolism were highly downregulated at both  <dig> and  <dig> min;  the genes of inorganic ion transport and metabolism were downregulated at  <dig> min and decrease in the number of genes downregulated at  <dig> min was also observed;  the genes in the class of "translation, ribosomal structure and biogenesis" were significantly upregulated at  <dig> min;  the number of genes involved in nucleotide transport and metabolism were increased after  <dig> min compared to after  <dig> min.
in general, figure  <dig> illustrates that the functional classes contained more downregulated and fewer upregulated genes at  <dig> min.
this result suggests that the functional class profiles were notably different between  <dig> and  <dig> min, and this difference might explain why s. aureus underwent the initial growth inhibition followed by partial growth recovery upon exposure to opp.
functional classifications analysis
to further identify genes with similar transcription patterns during the time course, we removed  <dig> genes , hypothetical protein  and general function predicted only ).
we categorized  <dig> genes with known functions into  <dig> groups on the basis of their transcription directions .
briefly, group i contained  <dig> genes upregulated upon both exposure times, while group ii had  <dig> genes with increased expression levels at  <dig> min and no significant changes upon  <dig> min exposure.
further, group iii possessed  <dig> genes that were upregulated at  <dig> min exposure.
group iv contained  <dig> genes downregulated upon both exposure times, whereas  <dig> genes of group v exhibited downregulation after  <dig> min.
finally, group vi had  <dig> genes that were downregulated upon  <dig> min exposure.
figure  <dig> displays the number of genes  within groups i through vi in each functional class.
as indicated above, additional file  <dig> contains all  <dig> genes including the genes classified under the functional group designated as "unknown, hypothetical, and general function prediction only".
since most of the genes discussed in this report are in additional file  <dig>  for further analysis of the data and for the readers convenience, we decided to make table  <dig> with  <dig> s. aureus genes that were most strongly upregulated or downregulated in response to opp after  <dig> and  <dig> minutes exposure.
these genes were also classified under seven groups based on their transcription directions
* genes were validated by real-time pcr.
group i: genes upregulated upon  <dig> and  <dig> min exposures
group i of table  <dig> contains  <dig> genes associated with virulence in s. aureus.
interestingly, five of these genes encode the secretory antigen precursor, ssaa.
the ssaa gene potentially regulated by the yycg/yycf system encodes proteins involved in cell wall metabolism, membrane-bound transport systems, and pathogenicity, including two major antigenic proteins, ssaa and isaa.
yycf has also been shown to bind specifically to the promoter regions of two genes, encoding the isaa antigen and the lytm peptidoglycan hydrolase.
this is in agreement with the proposed role of this system in controlling virulence and cell wall metabolism  <cit> .
in this study, opp also upregulated the clfb  gene expression upon both  <dig> and  <dig> min exposure.
in our previous results, triclosan upregulated the expression of sa <dig> encoding clfb, which binds fibrinogen  <cit> .
the results of this study show that the production of virulence factors in s. aureus may be a secondary effect of opp and this may provide new insight into the protective response of s. aureus to opp.
group ii: genes upregulated upon  <dig> min exposure
group ii of table  <dig> indicates that the class of "translation, ribosomal structure and biogenesis" which is responsible for the synthesis of ribosomal proteins was upregulated after  <dig> minutes.
in group ii of table  <dig>  for instance four genes encode 30s ribosomal proteins and  <dig> genes code for 50s ribosomal proteins.
in addition, sa <dig>  which encoded general stress protein was upregulated at  <dig> min.
ribosomal proteins are required for protein translation.
such early response of these ribosomal protein genes may reflect a stress response during exposure.
the upregulation of ribosomal protein genes might enhance the translation process or help proper ribosome functioning under stress conditions as exposure to opp.
the suggestion that the expression of ribosomal proteins is activated upon exposure to opp is surprising as this presumably reflects increased growth rate or virulence.
in group ii , we also observed genes related to primary metabolism that mainly belonged to the functional classes of "purines, pyrimidines, nucleosides, and nucleotides".
the gene cluster: sa1041–sa <dig>  which is homologous to the pyrimidine biosynthetic  operon of bacillus subtilis  <cit>  was upregulated at  <dig> min.
group iii: genes upregulated upon  <dig> min exposure
in group iii of table  <dig>  there were some genes from amino acid transport and metabolism, an atp-binding cassette  transporters and transcription.
the oligopeptide transport system  of s. aureus is an abc transporter that transports amino acids, cations- and iron-carrying compounds and peptides with a broad specificity  <cit> .
the peptides are mainly used as nutrients by the multiple amino acid auxotrophic s. aureus.
the opp system consists of four different proteins: oppb and oppc, oppd and oppf.
interestingly,  <dig> of the  <dig> genes, including sa0845–sa <dig> that code for proteins associated with amino acid transport were upregulated .
therefore, the suggestion that the expression of these proteins is activated upon exposure to opp is surprising as this presumably reflects increased growth rate or recovery.
we found that a putative operon containing four open reading frames   was upregulated .
the potabcd operon encodes a periplasmic binding protein dependent abc transport systems from gram-positive bacteria  <cit> .
the sa0950–sa0952–sa <dig> shows homology to the genes encoding this potabcd transport system , which are involved in the transport of spermidine and putrescine.
further, we showed the upregulation of abc transport systems-related genes, which accompanied the growth recovery.
of further importance was that group iii contained genes related to integral membrane protein, which belonged to the functional class of "cell division and chromosome partitioning".
sa <dig>  is a putative integral membrane protein possibly involved in chromosome condensation .
group iv: genes downregulated upon  <dig> and  <dig> min exposures
in group iv in table  <dig>  we noted that genes belonging to the functional classes of "amino acid transport and metabolism", "carbohydrate transport and metabolism", "energy production and conversion", "posttranslational modification protein turnover chaperones", "transcription" classes and putative lipoproteins were downregulated upon both exposure times.
intriguingly, we observed the high downregulation of sa <dig> and sa <dig> , the heme-regulated transport system, which consist of a novel transport system which plays a critical role in staphylococcal heme metabolism .
among the genes in the class of "amino acid transport and metabolism", sa <dig> -sa <dig> -sa <dig> -sa <dig> -sa <dig> , and sa <dig>  fall within a predicted operon and are all involved in diaminopimelate  biosynthesis .
the disruption of biosynthetic pathways involved in building up bacterial cell wall components is a common mode of action of antibiotics  <cit> .
penicillins  <cit> , methicillin  <cit> , cephalosporins  <cit>  and glycopeptide drugs such as vancomycin  <cit>  are all drugs that inhibit major steps in the construction of the peptidoglycan layer of bacterial cell walls.
lysine or its biosynthetic precursor, dap  <cit> , are essential to most bacteria for the synthesis of the peptidoglycan layer of the cell wall  <cit> .
since mammals neither make nor use dap and require l-lysine is an essential amino acid that is supplied through dietary intake, inhibitors of the dap biosynthetic pathway will probably not result in mammalian toxicity.
decisively sa <dig> -sa <dig> -sa <dig> -sa <dig>  and sa <dig>  show fold highest decreases as - <dig> , - <dig> , - <dig> , - <dig> , and - <dig>  folds at  <dig> min and - <dig> , - <dig> , - <dig> , - <dig> , and - <dig>  folds at  <dig> min in this experiment .
our findings suggest that the mode of action of opp may be related to bacterial biosynthesis of amino acids.
other genes of amino acids, including methionine, threonine, histidine and lysine were also highly down regulated at - <dig>  - <dig>  - <dig> and - <dig> folds.
therefore, this outcome in conjunction with the extensive downregulation of the genes encoding dap biosynthesis suggests that opp may inhibit construction of the peptidoglycan in cell wall of s. aureus.
these genes were less downregulated at  <dig> min than at  <dig> min.
these results suggest that opp inhibits the growth of s. aureus at  <dig> min and that growth recovery occurs at  <dig> minutes, indicating a possible mechanism of action of opp in s. aureus.
in a similar study carried out using pseudomonas aeruginosa treated with  <dig>  mm opp, we did not observe extensive downregulation of the genes involved in amino acid biosynthesis and specifically lysine biosynthesis .
this suggests that the mechanisms of action of  <dig>  mm opp on p. aeruginosa and s. aureus may differ.
the real time pcr results are the mean of three biological replicates with three technical replicates for each gene.
the microarray results are the mean of five replicates of each gene.
asa <dig> was glyceraldehyde 3-phosphate dehydrogenase  and used as the house-keeping gene.
bsa <dig>  sa <dig>  sa <dig>  and sa <dig> were downregulated at  <dig> min with no change at  <dig> min.
csa <dig>  sa <dig>  and sa <dig> were downregulated after  <dig> min and upregulated after  <dig> min exposure.
additional amino acid biosynthesis genes including: sa <dig> -sa <dig> -sa <dig>  involved in threonine biosynthesis were also in this group .
further, sa2082–sa2083–sa2084–sa2085–sa2086–sa <dig> , which make co <dig> and nh <dig> from urea and encode urea amidohydrolase and urease accessory proteins and sa <dig> which codes for serine-pyruvate aminotransferase were downregulated at  <dig> and  <dig> min .
group iv of table  <dig> also shows that the functional class of "cell envelope biogenesis, outer membrane" was distinctive.
in particular, sa <dig> which shows an -18-fold decrease after  <dig> min encodes an alanine racemase that catalyses the conversion of l-alanine into d-alanine, a key component of bacterial peptidoglycan  <cit> .
additionally, the putative lipoproteins: sa <dig> , sa <dig> -sa <dig> , sa <dig> , sa <dig> , and sa <dig> which are cell wall anchoring surface proteins were downregulated in response to opp .
in group iv, we also observed genes related to primary metabolism that mainly belonged to the functional classes of "energy metabolism", "lipid metabolism", and "transcription".
for example, cytochrome bd complex: sa0937–sa <dig>  was downregulated upon both  <dig> min and  <dig> min exposure .
cytochrome bd complex is one of two terminal oxidases in the bacterial respiratory chain that reduce molecular oxygen to water, utilizing intermediates shuttled through the electron transport chain  <cit> .
cytochrome d oxidase catalyses the last step of oxygen respiration and prevails under oxygen-limiting conditions  <cit> .
interestingly, it was speculated that cytochrome d oxidase is required under conditions of environmental stress and may have crucial roles in cellular physiology other than acting as an oxidase  <cit> .
however, prior studies revealed that the cydab genes were strongly upregulated upon exposure to hydrogen peroxide strengthens the confidence of the prior assignments about the role of cytochrome d oxidase in oxidative protection processes of both gram positive and gram negative bacteria  <cit> .
group v: genes downregulated upon  <dig> min exposures
in group v in table  <dig>  the most dominant class was "amino acid transport and metabolism", which contained half of the genes in that group.
further, sa <dig> -sa <dig>  and sa <dig> -sa <dig> -sa <dig> -sa <dig> -sa <dig> -sa <dig>  which were downregulated  are possibly parts of an operon homologous to the ilv-leu operon encoding enzymes of branched-chain amino acid biosynthesis in bacillus subtilis  <cit> .
in addition, sa <dig> -sa <dig> -sa2466–sa <dig> -sa <dig> -sa <dig> and sa <dig>  which are possibly parts of an operon homologous to the histidine biosynthesis were highly downregulated on  <dig> min .
sa1200–sa <dig> -sa <dig> -sa <dig> -sa <dig> -as <dig>  which are also possibly parts of an operon homologous to the tryptophan biosynthesis operon are downregulated on  <dig> min .
this result along with the downregulation of  <dig> genes involved in amino acid biosynthesis in group v suggests that amino acid synthesis was repressed upon  <dig> min exposure to opp in s. aureus.
group v shows that  <dig> genes in the functional class of "inorganic ion transport and metabolism" were downregulated at  <dig> min.
first, sa <dig>  codes for probable d-methionine-binding lipoprotein .
the proteins encoded by sa <dig> -sa <dig> -sa <dig> -sa <dig> -sa <dig>  are involved in d-methionine transporter of s. aureus abc transporter .
interestingly, group v contained lipoproteins such as sa <dig>  sa <dig>  sa <dig>  sa <dig>  sa <dig> and sa <dig> .
these results, along with downregulation of all the genes of lipoproteins of s. aureus in group iv and v, suggest that opp exposure may decrease stability of the staphylococcal membrane.
secondly, the proteins encoded by sa <dig> -sa <dig>  are involved in cobalt and nickel transport .
group vi: genes downregulated upon  <dig> min exposures
one of the characteristics of group vi of table  <dig> was the downregulation of  <dig> genes belonging to the functional class of "cell wall/membrane/envelope biogenesis".
in particular, genes related to envelope biogenesis were distinctive: sa <dig> -sa <dig> -sa <dig> -sa <dig> -sa <dig> -sa <dig> -sa <dig> -sa <dig> -sa <dig> -sa <dig>  were downregulated at  <dig> min.
these genes share homology with the capsular polysaccharide synthesis enzyme  operon which in turn is essential for virulence by impeding phagocytosis  <cit> .
this finding is congruent with the previous outcome that triclosan downregulates several virulence factor-related genes ) in s. aureus  <cit> .
moreover, isdac encoded by sa <dig> and sa <dig>  the iron-regulated surface determinant  system, was downregulated at  <dig> min .
identification of the isd system in s. aureus has demonstrated the importance of cell-wall sorted proteins in heme binding and transport  <cit> .
to date, the isd system comprises the only known heme-iron utilization pathway in s. aureus.
cell-wall sorted proteins of the s. aureus iron-regulated surface determinant system bind human hemoproteins, remove the heme molecule, and transport heme through the cell wall and plasma membrane for accumulation in the bacterial cytoplasm  <cit> .
particularly important was that many of the genes in the class of "coenzyme metabolism" were also members of group vi .
intriguingly, the genes were all involved in the riboflavin biosynthesis.
sa <dig> -sa <dig> -sa <dig> -sa <dig>  was downregulated at  <dig> min exposure .
riboflavin  is an essential component of the basic metabolism, being a precursor of coenzymes flavin adenine dinucleotide  and flavin mononucleotide .
the best studied system of the riboflavin biosynthesis in bacteria is the rib operon of bacillus subtilis encoding a pyrimidine deaminase/reductase, α-subunit of riboflavin synthase, gtp cyclohydrolase/ <dig> -dihydroxy 2-butanone 4-phosphate  synthase, and β-subunit of riboflavin synthase  <cit> .
these enzymes form a pathway that creates one riboflavin molecule from one molecule of gtp and two molecules of ribulose 5-phosphate  <cit> .
the proteins encoded by sa <dig> -sa <dig> -sa <dig>  were involved in thiamine biosynthesis of coenzyme metabolism at  <dig> min .
methicillin-resistant s. aureus small colony variants are frequently auxotrophic for hemin, menadione, thiamine, and co <dig> involved in biosynthesis of the electron transport chain element.
this phenotype grows slowly, and forms very small, nonhemolytic colonies in routine culture, so it may lead to the misidentification of this organism.
as discussed above, group iv also had sa <dig> -sa <dig> , which exhibited expression level decreases upon  <dig> and  <dig> min exposures in chorus with the repression of the genes of thiamine biosynthesis.
therefore, this result suggests that growth inhibition was accompanied with the repression of many coenzyme metabolism-related genes.
group vii: genes downregulated upon  <dig> min and upregulated upon  <dig> min exposures
note that group vii has been included only in table  <dig> in order to discuss the aberrant behaviour of the ica genes.
group vii is not indicated on figure  <dig> and  <dig>  intriguingly, we observed that sa <dig>  sa <dig> and sa <dig>  which make up the intercellular adhesion  operon and contribute to virulence in s. aureus were downregulated after  <dig> min and upregulated after  <dig> min of exposure to opp .
the intercellular adhesion operon  mediates polysaccharide intercellular adhesion in s. aureus, which leads to cell-cell adhesion and is required for biofilm formation  <cit>  prior studies have demonstrated that polysaccharide intercellular adhesin/hemagglutinin production is involved in the pathogenesis of s. epidermidis  <cit> , and is also upregulated by subinhibitory concentrations of certain antibiotics  <cit> .
therefore, this finding proposes that biofilm formation may not occur after  <dig> min of exposure to opp but is possibly favoured as a protective response as exposure time increases to  <dig> minutes.
validation of array data by real-time pcr
as an independent measure of differential gene expression, we examined the relative levels of  <dig> genes with a range of fold changes  by real-time pcr analysis, which were specifically involved in the pathogenesis or metabolism of s. aureus.
table  <dig> shows that our microarray results were corroborated with real-time pcr analysis, which provides independent verification of transcript level changes of the genes that we discuss in this report.
CONCLUSIONS
in this paper, we demonstrated how opp upregulated and downregulated genes in s. aureus, for the first time, by utilizing whole-genome microarrays.
moreover, we presented how the transcriptome profile of s. aureus was shifted during its cellular response to opp, which involved the growth inhibition.
to our knowledge, this is the first study demonstrating the activation of fermentative metabolism after opp treatment in s. aureus.
in summary, we revealed that amino acid metabolism genes were selectively downregulated between  <dig> and  <dig> minutes when exposed to  <dig>  mm opp.
we also found that the growth inhibition was accompanied by the downregulation of many membrane function-related genes; however, the majority of these genes returned to normal transcription levels during the growth resumption.
further, we showed that the repression of the iron-regulated surface determinant  system, hemin and thiamine-related genes accompanied with the growth inhibition.
notably, we discovered the upregulation of virulence genes and ribosomal genes while the cells returned to normal growth.
these results suggest that s. aureus might be arrested upon exposure to opp.
in this study, opp treatment led to the downregulation of several genes involved in amino acid anabolism.
the genes involved in the dap and lysine biosynthetic pathways were most significantly downregulated.
lysine and dap are essential for building up the peptidoglycan cell wall.
this finding proposes that the mode of action of the antimicrobial, opp in s. aureus might be attributed to the inhibition of genes of lysine biosynthesis and subsequently peptidoglycan biosynthesis.
we can therefore, conclude, that the mode of action of opp is similar to the mechanism of action of some antibiotics.
this study has revealed novel information on the mechanism of action of opp in s. aureus which will benefit further antimicrobial research on s. aureus.

 the u.s. environmental protection agency has endeavored to determine the efficacy and the mode of action of antimicrobials. at epa, antimicrobial products are registered, and hospital-level disinfectants are being tested against pathogens such as s. aureus, which is responsible for many infectious diseases, ranging from benign skin infections to life-threatening endocarditis and toxic shock syndrome. one of the reasons epa has exerted such efforts is that hospital-acquired infections are a serious threat to public health. therefore, it is important to use appropriate antimicrobial agents with clear understanding of the subsequent effects to prevent infection outbreaks in health care environments. the phenolic compound, ortho-phenylphenol, is an antimicrobial agent and an active ingredient of epa-registered disinfectant with wide human exposure in various agricultural, hospital and veterinary disinfectant products. opp is employed in a variety of applications, including hard surface disinfection, wood preservation, treatment of citrus fruit, vegetables before packaging to prevent microbial decay and textile production due to its bactericidal and fungicidal activity. there have been several reports related to the exposure of opp on humans. it has been reported that opp increased the incidence of urinary bladder tumors in f344/ducrj rats when administered in the diet. the results of this study stimulated the initiation of additional testing of opp for both tumor induction and possible reactivity with dna. opp has been found to have estrogenic or anti-androgenic activity, and binds to the androgen or estrogen receptors. in spite of these effects opp is still used in applications that simultaneously contact both humans and bacteria. it is therefore important to understand the differential effects on each so that its efficacy can be understood and even optimized. moreover, a lack of understanding of a cellular response to opp hinders further development of more innovative methods for combating pathogens. certainly, better elucidation of the molecular events responsible for establishing and maintaining pathogenicity will help to map affected cell functions and serve to delineate the mechanisms involved in the disinfectant activity. microarrays have been effectively employed to simultaneously and globally examine the complete transcriptional response at the genomic level in pseudomonas aeruginosa and s. aureus upon exposure to antimicrobials. in this study, to our knowledge, for the first time, we show that the global transcription response of s. aureus to opp includes downregulation of genes involved in lysine metabolism, as well as genes involved in amino acid metabolism, by utilizing affymetrix s. aureus genechip arrays. our findings indicate that: many cellular protective processes were upregulated, the transcription of genes involved in primary metabolic pathways was downregulated, and the transcription of genes encoding lysine and histidine biosynthesis was downregulated. next we performed real-time pcr analysis on selected genes to validate the array results. based on this result, it was concluded that this study may help to elucidate the mechanism of action by which opp stops cell wall construction and thereby inhibiting s. aureus growth, and may facilitate the design of more effective antimicrobials.  growth inhibition by opp to determine the sublethal inhibitory effect of opp on s. aureus, we first exposed the exponentially growing cells to different concentrations of opp dissolved in dmso. in figure we demonstrate that mm concentration of opp caused a growth inhibition for about min. note that minimum inhibitory concentration of opp on s. aureus was reportedly mg/l. in this study, to better understand how s. aureus initially responds to opp, we chose the rate of cell growth inhibition with mm opp after and min exposure times compared to control. transcriptional profiles in response to opp to investigate early transcriptional changes in response to opp exposure, we isolated total rna after min and minutes exposure to mm opp and conducted five independent microarray experiments in the absence and the presence of mm opp. to further identify genes with statistically marked changes in expression levels, we applied the following criteria to each of the min, min, and control-experimental microarray data sets: a p-value for a t-test should be equal to or less than, an absolute fold change in transcript level should be equal to or greater than and a gene should have a presence or marginal call from 50% or more replicates on both the experimental and control replicate sets. of the genes represented on the s. aureus genechip, genes showed statistical significance based on a 1-way anova. we found that mrna levels genes of s. aureus were significantly altered in response to opp by two fold or more upregulation or downregulation. the raw data of genes control and experimental genes after has been deposited in ncbi's gene expression omnibus and is accessible through geo series accession number gse. analysis of gene expression changes in min and min to examine how genes with transcript level changes are distributed with regard to their functions, we further classified these genes that were either upregulated or downregulated by a fold change of two or more according to the gene classification based on cog functional categories in the genome of national center for biotechnology information. in figure the differences between the numbers of up and downregulated genes in each functional class after and minutes exposure to mm of opp are illustrated. note that figure represents a total of genes including the group of "function unknown, hypothetical protein and general function predicted only ". some interesting findings are as follows: the genes of amino acid transport and metabolism were highly downregulated at both and min; the genes of inorganic ion transport and metabolism were downregulated at min and decrease in the number of genes downregulated at min was also observed; the genes in the class of "translation, ribosomal structure and biogenesis" were significantly upregulated at min; the number of genes involved in nucleotide transport and metabolism were increased after min compared to after min. in general, figure illustrates that the functional classes contained more downregulated and fewer upregulated genes at min. this result suggests that the functional class profiles were notably different between and min, and this difference might explain why s. aureus underwent the initial growth inhibition followed by partial growth recovery upon exposure to opp. functional classifications analysis to further identify genes with similar transcription patterns during the time course, we removed genes, hypothetical protein and general function predicted only ). we categorized genes with known functions into groups on the basis of their transcription directions. briefly, group i contained genes upregulated upon both exposure times, while group ii had genes with increased expression levels at min and no significant changes upon min exposure. further, group iii possessed genes that were upregulated at min exposure. group iv contained genes downregulated upon both exposure times, whereas genes of group v exhibited downregulation after min. finally, group vi had genes that were downregulated upon min exposure. figure displays the number of genes within groups i through vi in each functional class. as indicated above, additional file contains all genes including the genes classified under the functional group designated as "unknown, hypothetical, and general function prediction only". since most of the genes discussed in this report are in additional file for further analysis of the data and for the readers convenience, we decided to make table with s. aureus genes that were most strongly upregulated or downregulated in response to opp after and minutes exposure. these genes were also classified under seven groups based on their transcription directions * genes were validated by real-time pcr. group i: genes upregulated upon and min exposures group i of table contains genes associated with virulence in s. aureus. interestingly, five of these genes encode the secretory antigen precursor, ssaa. the ssaa gene potentially regulated by the yycg/yycf system encodes proteins involved in cell wall metabolism, membrane-bound transport systems, and pathogenicity, including two major antigenic proteins, ssaa and isaa. yycf has also been shown to bind specifically to the promoter regions of two genes, encoding the isaa antigen and the lytm peptidoglycan hydrolase. this is in agreement with the proposed role of this system in controlling virulence and cell wall metabolism. in this study, opp also upregulated the clfb gene expression upon both and min exposure. in our previous results, triclosan upregulated the expression of sa encoding clfb, which binds fibrinogen. the results of this study show that the production of virulence factors in s. aureus may be a secondary effect of opp and this may provide new insight into the protective response of s. aureus to opp. group ii: genes upregulated upon min exposure group ii of table indicates that the class of "translation, ribosomal structure and biogenesis" which is responsible for the synthesis of ribosomal proteins was upregulated after minutes. in group ii of table for instance four genes encode 30s ribosomal proteins and genes code for 50s ribosomal proteins. in addition, sa which encoded general stress protein was upregulated at min. ribosomal proteins are required for protein translation. such early response of these ribosomal protein genes may reflect a stress response during exposure. the upregulation of ribosomal protein genes might enhance the translation process or help proper ribosome functioning under stress conditions as exposure to opp. the suggestion that the expression of ribosomal proteins is activated upon exposure to opp is surprising as this presumably reflects increased growth rate or virulence. in group ii, we also observed genes related to primary metabolism that mainly belonged to the functional classes of "purines, pyrimidines, nucleosides, and nucleotides". the gene cluster: sa1041–sa which is homologous to the pyrimidine biosynthetic operon of bacillus subtilis was upregulated at min. group iii: genes upregulated upon min exposure in group iii of table there were some genes from amino acid transport and metabolism, an atp-binding cassette transporters and transcription. the oligopeptide transport system of s. aureus is an abc transporter that transports amino acids, cations- and iron-carrying compounds and peptides with a broad specificity. the peptides are mainly used as nutrients by the multiple amino acid auxotrophic s. aureus. the opp system consists of four different proteins: oppb and oppc, oppd and oppf. interestingly, of the genes, including sa0845–sa that code for proteins associated with amino acid transport were upregulated. therefore, the suggestion that the expression of these proteins is activated upon exposure to opp is surprising as this presumably reflects increased growth rate or recovery. we found that a putative operon containing four open reading frames was upregulated. the potabcd operon encodes a periplasmic binding protein dependent abc transport systems from gram-positive bacteria. the sa0950–sa0952–sa shows homology to the genes encoding this potabcd transport system, which are involved in the transport of spermidine and putrescine. further, we showed the upregulation of abc transport systems-related genes, which accompanied the growth recovery. of further importance was that group iii contained genes related to integral membrane protein, which belonged to the functional class of "cell division and chromosome partitioning". sa is a putative integral membrane protein possibly involved in chromosome condensation. group iv: genes downregulated upon and min exposures in group iv in table we noted that genes belonging to the functional classes of "amino acid transport and metabolism", "carbohydrate transport and metabolism", "energy production and conversion", "posttranslational modification protein turnover chaperones", "transcription" classes and putative lipoproteins were downregulated upon both exposure times. intriguingly, we observed the high downregulation of sa and sa, the heme-regulated transport system, which consist of a novel transport system which plays a critical role in staphylococcal heme metabolism. among the genes in the class of "amino acid transport and metabolism", sa -sa -sa -sa -sa, and sa fall within a predicted operon and are all involved in diaminopimelate biosynthesis. the disruption of biosynthetic pathways involved in building up bacterial cell wall components is a common mode of action of antibiotics. penicillins, methicillin, cephalosporins and glycopeptide drugs such as vancomycin are all drugs that inhibit major steps in the construction of the peptidoglycan layer of bacterial cell walls. lysine or its biosynthetic precursor, dap, are essential to most bacteria for the synthesis of the peptidoglycan layer of the cell wall. since mammals neither make nor use dap and require l-lysine is an essential amino acid that is supplied through dietary intake, inhibitors of the dap biosynthetic pathway will probably not result in mammalian toxicity. decisively sa -sa -sa -sa and sa show fold highest decreases as -, -, -, -, and - folds at min and -, -, -, -, and - folds at min in this experiment. our findings suggest that the mode of action of opp may be related to bacterial biosynthesis of amino acids. other genes of amino acids, including methionine, threonine, histidine and lysine were also highly down regulated at - - - and - folds. therefore, this outcome in conjunction with the extensive downregulation of the genes encoding dap biosynthesis suggests that opp may inhibit construction of the peptidoglycan in cell wall of s. aureus. these genes were less downregulated at min than at min. these results suggest that opp inhibits the growth of s. aureus at min and that growth recovery occurs at minutes, indicating a possible mechanism of action of opp in s. aureus. in a similar study carried out using pseudomonas aeruginosa treated with mm opp, we did not observe extensive downregulation of the genes involved in amino acid biosynthesis and specifically lysine biosynthesis. this suggests that the mechanisms of action of mm opp on p. aeruginosa and s. aureus may differ. the real time pcr results are the mean of three biological replicates with three technical replicates for each gene. the microarray results are the mean of five replicates of each gene. asa was glyceraldehyde 3-phosphate dehydrogenase and used as the house-keeping gene. bsa sa sa and sa were downregulated at min with no change at min. csa sa and sa were downregulated after min and upregulated after min exposure. additional amino acid biosynthesis genes including: sa -sa -sa involved in threonine biosynthesis were also in this group. further, sa2082–sa2083–sa2084–sa2085–sa2086–sa, which make co and nh from urea and encode urea amidohydrolase and urease accessory proteins and sa which codes for serine-pyruvate aminotransferase were downregulated at and min. group iv of table also shows that the functional class of "cell envelope biogenesis, outer membrane" was distinctive. in particular, sa which shows an -18-fold decrease after min encodes an alanine racemase that catalyses the conversion of l-alanine into d-alanine, a key component of bacterial peptidoglycan. additionally, the putative lipoproteins: sa, sa -sa, sa, sa, and sa which are cell wall anchoring surface proteins were downregulated in response to opp. in group iv, we also observed genes related to primary metabolism that mainly belonged to the functional classes of "energy metabolism", "lipid metabolism", and "transcription". for example, cytochrome bd complex: sa0937–sa was downregulated upon both min and min exposure. cytochrome bd complex is one of two terminal oxidases in the bacterial respiratory chain that reduce molecular oxygen to water, utilizing intermediates shuttled through the electron transport chain. cytochrome d oxidase catalyses the last step of oxygen respiration and prevails under oxygen-limiting conditions. interestingly, it was speculated that cytochrome d oxidase is required under conditions of environmental stress and may have crucial roles in cellular physiology other than acting as an oxidase. however, prior studies revealed that the cydab genes were strongly upregulated upon exposure to hydrogen peroxide strengthens the confidence of the prior assignments about the role of cytochrome d oxidase in oxidative protection processes of both gram positive and gram negative bacteria. group v: genes downregulated upon min exposures in group v in table the most dominant class was "amino acid transport and metabolism", which contained half of the genes in that group. further, sa -sa and sa -sa -sa -sa -sa -sa which were downregulated are possibly parts of an operon homologous to the ilv-leu operon encoding enzymes of branched-chain amino acid biosynthesis in bacillus subtilis. in addition, sa -sa -sa2466–sa -sa -sa and sa which are possibly parts of an operon homologous to the histidine biosynthesis were highly downregulated on min. sa1200–sa -sa -sa -sa -as which are also possibly parts of an operon homologous to the tryptophan biosynthesis operon are downregulated on min. this result along with the downregulation of genes involved in amino acid biosynthesis in group v suggests that amino acid synthesis was repressed upon min exposure to opp in s. aureus. group v shows that genes in the functional class of "inorganic ion transport and metabolism" were downregulated at min. first, sa codes for probable d-methionine-binding lipoprotein. the proteins encoded by sa -sa -sa -sa -sa are involved in d-methionine transporter of s. aureus abc transporter. interestingly, group v contained lipoproteins such as sa sa sa sa sa and sa. these results, along with downregulation of all the genes of lipoproteins of s. aureus in group iv and v, suggest that opp exposure may decrease stability of the staphylococcal membrane. secondly, the proteins encoded by sa -sa are involved in cobalt and nickel transport. group vi: genes downregulated upon min exposures one of the characteristics of group vi of table was the downregulation of genes belonging to the functional class of "cell wall/membrane/envelope biogenesis". in particular, genes related to envelope biogenesis were distinctive: sa -sa -sa -sa -sa -sa -sa -sa -sa -sa were downregulated at min. these genes share homology with the capsular polysaccharide synthesis enzyme operon which in turn is essential for virulence by impeding phagocytosis. this finding is congruent with the previous outcome that triclosan downregulates several virulence factor-related genes ) in s. aureus. moreover, isdac encoded by sa and sa the iron-regulated surface determinant system, was downregulated at min. identification of the isd system in s. aureus has demonstrated the importance of cell-wall sorted proteins in heme binding and transport. to date, the isd system comprises the only known heme-iron utilization pathway in s. aureus. cell-wall sorted proteins of the s. aureus iron-regulated surface determinant system bind human hemoproteins, remove the heme molecule, and transport heme through the cell wall and plasma membrane for accumulation in the bacterial cytoplasm. particularly important was that many of the genes in the class of "coenzyme metabolism" were also members of group vi. intriguingly, the genes were all involved in the riboflavin biosynthesis. sa -sa -sa -sa was downregulated at min exposure. riboflavin is an essential component of the basic metabolism, being a precursor of coenzymes flavin adenine dinucleotide and flavin mononucleotide. the best studied system of the riboflavin biosynthesis in bacteria is the rib operon of bacillus subtilis encoding a pyrimidine deaminase/reductase, α-subunit of riboflavin synthase, gtp cyclohydrolase/ -dihydroxy 2-butanone 4-phosphate synthase, and β-subunit of riboflavin synthase. these enzymes form a pathway that creates one riboflavin molecule from one molecule of gtp and two molecules of ribulose 5-phosphate. the proteins encoded by sa -sa -sa were involved in thiamine biosynthesis of coenzyme metabolism at min. methicillin-resistant s. aureus small colony variants are frequently auxotrophic for hemin, menadione, thiamine, and co involved in biosynthesis of the electron transport chain element. this phenotype grows slowly, and forms very small, nonhemolytic colonies in routine culture, so it may lead to the misidentification of this organism. as discussed above, group iv also had sa -sa, which exhibited expression level decreases upon and min exposures in chorus with the repression of the genes of thiamine biosynthesis. therefore, this result suggests that growth inhibition was accompanied with the repression of many coenzyme metabolism-related genes. group vii: genes downregulated upon min and upregulated upon min exposures note that group vii has been included only in table in order to discuss the aberrant behaviour of the ica genes. group vii is not indicated on figure and intriguingly, we observed that sa sa and sa which make up the intercellular adhesion operon and contribute to virulence in s. aureus were downregulated after min and upregulated after min of exposure to opp. the intercellular adhesion operon mediates polysaccharide intercellular adhesion in s. aureus, which leads to cell-cell adhesion and is required for biofilm formation prior studies have demonstrated that polysaccharide intercellular adhesin/hemagglutinin production is involved in the pathogenesis of s. epidermidis, and is also upregulated by subinhibitory concentrations of certain antibiotics. therefore, this finding proposes that biofilm formation may not occur after min of exposure to opp but is possibly favoured as a protective response as exposure time increases to minutes. validation of array data by real-time pcr as an independent measure of differential gene expression, we examined the relative levels of genes with a range of fold changes by real-time pcr analysis, which were specifically involved in the pathogenesis or metabolism of s. aureus. table shows that our microarray results were corroborated with real-time pcr analysis, which provides independent verification of transcript level changes of the genes that we discuss in this report.  in this paper, we demonstrated how opp upregulated and downregulated genes in s. aureus, for the first time, by utilizing whole-genome microarrays. moreover, we presented how the transcriptome profile of s. aureus was shifted during its cellular response to opp, which involved the growth inhibition. to our knowledge, this is the first study demonstrating the activation of fermentative metabolism after opp treatment in s. aureus. in summary, we revealed that amino acid metabolism genes were selectively downregulated between and minutes when exposed to mm opp. we also found that the growth inhibition was accompanied by the downregulation of many membrane function-related genes; however, the majority of these genes returned to normal transcription levels during the growth resumption. further, we showed that the repression of the iron-regulated surface determinant system, hemin and thiamine-related genes accompanied with the growth inhibition. notably, we discovered the upregulation of virulence genes and ribosomal genes while the cells returned to normal growth. these results suggest that s. aureus might be arrested upon exposure to opp. in this study, opp treatment led to the downregulation of several genes involved in amino acid anabolism. the genes involved in the dap and lysine biosynthetic pathways were most significantly downregulated. lysine and dap are essential for building up the peptidoglycan cell wall. this finding proposes that the mode of action of the antimicrobial, opp in s. aureus might be attributed to the inhibition of genes of lysine biosynthesis and subsequently peptidoglycan biosynthesis. we can therefore, conclude, that the mode of action of opp is similar to the mechanism of action of some antibiotics. this study has revealed novel information on the mechanism of action of opp in s. aureus which will benefit further antimicrobial research on s. aureus. 
by using microarray analysis that enables us to simultaneously and globally examine the complete transcriptome during cellular responses, we have revealed novel information regarding the mode of action of opp on staphylococcus: opp inhibits anabolism of many amino acids and highly downregulates the genes that encode the enzymes involved in the dap pathway. intriguingly, we revealed that the transcription of genes encoding ribosomal proteins was upregulated by opp and at the same time, the genes encoding iron acquisition and transport were downregulated. in particular, the genes encoding the enzymes of the diaminopimelate pathway which results in lysine biosynthesis were significantly downregualted. staphylococcus aureus, is responsible for many infectious diseases, ranging from benign skin infections to life-threatening endocarditis and toxic shock syndrome. ortho-phenylphenol is an antimicrobial agent and an active ingredient of epa-registered disinfectants with wide human exposure in various agricultural, hospital and veterinary disinfectant products. our data indicated that opp downregulated the biosynthesis of many amino acids, which are required for protein synthesis. lysine and dap are essential for building up the peptidoglycan cell wall. despite many uses, an understanding of a cellular response to opp and it's mechanism of action, targeted genes, and the connectivity between targeted genes and the rest of cell metabolism remains obscure. herein, we performed a genome-wide transcriptome analysis of the cellular responses of s. aureus when exposed to mm of opp for and min. the discovery of this phenomenon provides useful information that will benefit further antimicrobial research on s. aureus. the genes encoding virulence factors were upregulated and genes encoding phospholipids were downregulated upon min exposure to opp.
 staphylococcus aureus, is responsible for many infectious diseases, ranging from benign skin infections to life-threatening endocarditis and toxic shock syndrome. ortho-phenylphenol is an antimicrobial agent and an active ingredient of epa-registered disinfectants with wide human exposure in various agricultural, hospital and veterinary disinfectant products. despite many uses, an understanding of a cellular response to opp and it's mechanism of action, targeted genes, and the connectivity between targeted genes and the rest of cell metabolism remains obscure.  herein, we performed a genome-wide transcriptome analysis of the cellular responses of s. aureus when exposed to mm of opp for and min. our data indicated that opp downregulated the biosynthesis of many amino acids, which are required for protein synthesis. in particular, the genes encoding the enzymes of the diaminopimelate pathway which results in lysine biosynthesis were significantly downregualted. intriguingly, we revealed that the transcription of genes encoding ribosomal proteins was upregulated by opp and at the same time, the genes encoding iron acquisition and transport were downregulated. the genes encoding virulence factors were upregulated and genes encoding phospholipids were downregulated upon min exposure to opp.  by using microarray analysis that enables us to simultaneously and globally examine the complete transcriptome during cellular responses, we have revealed novel information regarding the mode of action of opp on staphylococcus: opp inhibits anabolism of many amino acids and highly downregulates the genes that encode the enzymes involved in the dap pathway. lysine and dap are essential for building up the peptidoglycan cell wall. it was concluded that the mode of action of opp is similar to the mechanism of action of some antibiotics. the discovery of this phenomenon provides useful information that will benefit further antimicrobial research on s. aureus. 