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Antimicrob Agents Chemother. Feb 2012; 56(2): 787–804.
PMCID: PMC3264241

Global Analysis of the Staphylococcus aureus Response to Mupirocin


In the present study, we analyzed the response of S. aureus to mupirocin, the drug of choice for nasal decolonization. Mupirocin selectively inhibits the bacterial isoleucyl-tRNA synthetase (IleRS), leading to the accumulation of uncharged isoleucyl-tRNA and eventually the synthesis of (p)ppGpp. The alarmone (p)ppGpp induces the stringent response, an important global transcriptional and translational control mechanism that allows bacteria to adapt to nutritional deprivation. To identify proteins with an altered synthesis pattern in response to mupirocin treatment, we used the highly sensitive 2-dimensional gel electrophoresis technique in combination with mass spectrometry. The results were complemented by DNA microarray, Northern blot, and metabolome analyses. Whereas expression of genes involved in nucleotide biosynthesis, DNA metabolism, energy metabolism, and translation was significantly downregulated, expression of isoleucyl-tRNA synthetase, the branched-chain amino acid pathway, and genes with functions in oxidative-stress resistance (ahpC and katA) and putative roles in stress protection (the yvyD homologue SACOL0815 and SACOL1759 and SACOL2131) and transport processes was increased. A comparison of the regulated genes to known regulons suggests the involvement of the global regulators CodY and SigB in shaping the response of S. aureus to mupirocin. Of particular interest was the induced transcription of genes encoding virulence-associated regulators (i.e., arlRS, saeRS, sarA, sarR, sarS, and sigB), as well as genes directly involved in the virulence of S. aureus (i.e., fnbA, epiE, epiG, and seb).


Staphylococcus aureus is both a colonizer and a human pathogen. Due to its ability to express an impressive arsenal of different virulence factors, S. aureus is able to cause a variety of diseases ranging from mild skin infections to more severe and life-threatening conditions, such as osteomyelitis, endocarditis, and sepsis. With the emergence of multiresistant strains, antimicrobial therapy of S. aureus infections has become an increasing challenge.

Mupirocin (pseudomonic acid A) was first isolated from Pseudomonas fluorescens NCIB 10586 and has since proven its value as a topical agent in the treatment of S. aureus-associated wound infections. Furthermore, recent studies have shown that nasal decolonization of S. aureus carriers significantly reduces the risk of S. aureus infections after surgery (16, 28). However, the emergence of resistance to mupirocin after common use has increasingly hampered its use as the topical agent of choice for the elimination of methicillin-resistant Staphylococcus aureus (MRSA) carriage (6, 37, 75, 100, 112, 119).

Although the chemical structure of mupirocin is significantly different from that of isoleucyl-adenylate (Ile-AMP) (23), it was demonstrated that it is a structural analogue of Ile-AMP and competes with Ile-AMP for overlapping binding sites of the eubacterial and archaeal isoleucyl-tRNA synthetases (IleRSs) (66, 67, 91, 120). As a consequence, addition of mupirocin blocks the charging of isoleucyl-tRNA with isoleucine, leading to depletion of the aminoacetylated isoleucyl-tRNA pool and hence accumulation of uncharged isoleucyl-tRNAs.

The increase in uncharged tRNAs in relation to charged tRNAs is a signal of amino acid starvation for the cell, which triggers the stringent response. The stringent response is a fundamental global adaptation program universally distributed in most eubacteria and even partially conserved in the plastids of higher plants (99, 110). The major function of this response is to adjust the cell's biosynthetic machinery to the availability of energy and required precursors (59, 60, 103). The molecular details of the stringent response have been extensively studied in the model Gram-negative organism Escherichia coli and will be only briefly summarized here. Uncharged tRNAs that enter the ribosomal A site lead to a block of the ribosome and consequently protein synthesis (66). The arrest of the ribosome is recognized by the ribosome-associated RelA protein, which catalyzes the formation of the alarmones GTP 3′-diphosphate (pppGpp) and GDP 3′-diphosphate (ppGpp) from ATP as the phosphate donor and GTP or GDP (20, 57). In a DksA-dependent reaction, ppGpp is able to bind the RNA polymerase (RNAP) in E. coli, thereby blocking rRNA and tRNA gene transcription (5, 25, 113). Moreover, cell division and initiation of DNA synthesis are inhibited.

Stringently controlled promoters are often characterized by a specific discriminator region in front of the transcriptional start site. Whereas positively regulated genes are generally preceded by a CG-rich discriminator, negatively controlled genes have discriminator regions rich in adenine and thymidine nucleotides (99, 109, 114). Altogether, this course of events enables significant conservation of energy under growth-limiting conditions. However, transcription of amino acid biosynthesis and transport operons, as well as catabolic operons that yield precursors for amino acid synthesis, is enhanced in order to restore conditions that allow survival and growth (21, 93). Finally, (p)ppGpp is degraded by the hydrolase activity of SpoT. SpoT is a bifunctional enzyme that, in addition to its hydrolase activity, also has a (p)ppGpp synthetase activity that is activated by a variety of stimuli, such as inhibition of fatty acid metabolism or carbon deprivation.

Nevertheless, there are several general differences between the stringent responses of Gram-positive and Gram-negative bacteria (118). In most Gram-positive bacteria, (p)ppGpp synthetase and hydrolase activities are combined in a single protein, a bifunctional Rel/SpoT homolog (Rsh). In addition, a DksA homolog is absent from the genomes of firmicutes, such as Bacillus subtilis and S. aureus. Recently, in firmicutes, further proteins with (p)ppGpp synthetase activity have been discovered (92). The most striking differences, however, are related to the effector molecule. While in E. coli ppGpp directly affects RNAP activity, in B. subtilis the drop in the intracellular GTP pool as the result of the alarmone synthesis represents the virtual effector molecule. Interestingly, in B. subtilis, rRNA gene transcription starts predominantly with a guanosine nucleotide and can thus be affected by the intracellular GTP pool (72). In addition, GTP acts as a corepressor of the global stationary-phase regulator CodY, which also adapts downstream gene expression to stringent conditions (108).

Interestingly, in a number of pathogenic bacteria, including S. aureus, Mycobacterium tuberculosis, Listeria monocytogenes, and Pseudomonas aeruginosa, a connection between virulence and the stringent response has been observed (for a review, see references 30, 49, and 68).

Here, we investigated the response of S. aureus COL grown in a chemically defined medium in the presence of subinhibitory mupirocin concentrations by a combination of transcriptome, proteome, and metabolome analyses. The hallmark was the rapid and strong downregulation of the translation machinery and genes required for replication, as it is characteristic of the stringent response. Furthermore, the data suggested that global regulators, such as CodY, SigB, and possibly chromosomally encoded toxin/antitoxin modules contribute to the observed changes in gene expression. Surprisingly, many intracellular key metabolites showed little variation following mupirocin stress. A comparison to previous studies done in S. aureus and B. subtilis highlighted several poorly characterized but highly conserved genes that likely play pivotal roles during the stringent response. This functional-genomics approach provides a basis for more detailed experiments aimed at the dissection of the molecular mechanisms that govern the response of S. aureus to mupirocin treatment and the adaptation to stringent conditions.


Bacterial strain and growth conditions.

The S. aureus strain COL used in this study (104) was routinely grown aerobically in a defined synthetic medium (48) with vigorous agitation at 37°C. In order to determine the MIC of mupirocin, 5 ml of synthetic medium supplemented with increasing mupirocin (AppliChem, Darmstadt, Germany) concentrations was inoculated with 10 μl of exponentially growing S. aureus cells (optical density at 500 nm [OD500], 0.5) previously propagated in the same medium without antibiotics. The minimal mupirocin concentration that prevented turbidity of the medium after 14 h of culture was deemed the mupirocin MIC. Following this procedure, the mupirocin MIC for S. aureus COL was 0.01 μg/ml.

For mupirocin experiments, prewarmed medium was inoculated with cells from an exponentially growing overnight culture to an initial OD500 of 0.1 and monitored by measuring the OD until the culture reached an OD500 of 0.5. At that time, the culture was split, and one part was transferred to a prewarmed Erlenmeyer flask and treated with mupirocin while the remaining culture served as a control. The final mupirocin concentration used in all stress experiments was 0.03 μg/ml (3 times the mupirocin MIC), reducing the growth rate of S. aureus COL by 50%.

l-[35S]methionine labeling and preparation of pulse-labeled protein extracts.

In order to investigate changes in the pattern of proteins newly synthesized after the addition of mupirocin, the proteins were pulse-labeled with l-[35S]methionine as described previously (38). Briefly, at time points 10, 30, and 60 min after mupirocin treatment, 150 μCi of l-[35S]methionine was added to 10 ml of the respective cell culture. As a control, we pulse-labeled cells of an untreated culture at time point zero (exponential growth at an OD500 of 0.5, immediately before stress) and 60 min after the beginning of the stress experiment. The uptake and incorporation of radioactively labeled methionine in newly synthesized proteins always lasted exactly 5 min. Protein synthesis was blocked by the addition of 1 ml stop solution (0.1% [wt/vol] chloramphenicol, 10 mM unlabeled l-methionine, 0.1 M Tris-HCl [pH 7.5]). Cells were harvested on ice, centrifuged for 5 min (21,500 × g at 4°C), washed twice with ice-cold Tris-EDTA (TE) buffer (10 mM Tris-HCl [pH 7.7], 1 mM EDTA), and stored at −20°C. For the preparation of intracellular-protein extracts, cell pellets were resuspended in 400 μl of TE buffer containing lysostaphin (final concentration, 250 μg/ml), followed by incubation on ice for 10 min and disruption by sonication for 1 min (0.5 pulses per second; low), followed by a 1-min cooling break. This process was repeated three times. The cell lysate obtained was cleared by two centrifugation steps for 10 min and 40 min (21,500 × g at 4°C). The protein concentration was determined by using Roti-Nanoquant reagent according to the manufacturer's instructions (Roth, Karlsruhe, Germany), and the protein solutions were stored at −20°C.

To measure the incorporation of l-[35S]methionine into proteins, two aliquots of each protein solution (5 μl) was pipetted onto filter paper disks. After drying, they were subjected to 10 min of precipitation with 10% (wt/vol) trichloroacetic acid (TCA) on ice and washed for 10 min with 5% (wt/vol) TCA on ice and for 10 min with 90% (vol/vol) ethanol at room temperature. After drying, 1 ml toluene was added, and the radioactivity of the dried filter disks was measured as counts per minute on a Packard Tricarb 2900 TR liquid scintillation counter (PerkinElmer, Waltham, MA).

Preparation of cytoplasmic proteins for preparative two-dimen-sional (2D) gel electrophoresis.

Cells were separated from the supernatant by centrifugation (9,000 × g at 4°C) for 10 min, washed three times with ice-cold TE buffer, and resuspended in 1 ml ice-cold TE buffer. For cell lysis, 500 μl glass beads (0.01 to 0.1 mm) was added to the cell suspension, and the cells were disrupted by homogenization using a Ribolyser (Hybaid, United Kingdom) for 20 s at 6.5 m/s2 followed by incubation on ice for 4 min and a second disruption step for 30 s at the same acceleration. In order to remove cell debris and insoluble or aggregated proteins, the lysate was centrifuged for 25 min (21,500 × g at 4°C). The supernatant was transferred to a new tube, and the centrifugation step was repeated for 45 min. The protein concentration was determined as described above. Protein solutions were stored at −20°C.

Analytical and preparative 2D PAGE.

2D polyacrylamide gel electrophoresis (PAGE) was performed by using the immobilized pH gradient (IPG) technique as described previously (12). In the first dimension, proteins were separated on IPG strips (GE-Healthcare, Uppsala, Sweden) in a linear pH range of 4 to 7. For analytical 2D PAGE, 80 μg of radioactively labeled protein extracts was loaded onto the IPG strips. 2D PAGE analyses were performed as described previously (38). The resulting 2D gels were fixed with 50% (vol/vol) ethanol, 12% (vol/vol) acetic acid, and 500 μl/liter formaldehyde (37% [vol/vol]) for 1 to 2 h and subsequently stained with silver nitrate (15). The stained gels were scanned, dried in a vacuum dryer, and fixed onto Whatman paper. Afterward, the dried gels were exposed to storage phosphor screens (Molecular Dynamics, Buckinghamshire, United Kingdom) and scanned with a Typhoon scanner 9400 (Amersham Biosciences, Little Chalfont, United Kingdom) until the signal intensity reached a maximum value between 90,000 and 100,000.

For the identification of protein spots by mass spectrometry (MS), preparative 2D PAGE was performed. A total amount of 350 μg of protein extracts was loaded onto the IPG strips (GE Healthcare, Uppsala, Sweden) in a pH range of 4 to 7. The 2D gels obtained were fixed with 40% (vol/vol) ethanol and 10% (vol/vol) acetic acid for 1 h and then immediately stained with colloidal Coomassie brilliant blue (19). The gels were scanned with a Scanner X finity Ultra (Quato Graphic, Braunschweig, Germany).

Protein quantitation approaches.

For quantitation of cytoplasmic proteins, the 2D gel image analysis was performed with the software DELTA 2D version 3.4.1 beta 24 (Decodon GmbH, Greifswald, Germany). For each sampling point, gel images generated from two biological replicates were used to create a virtual fusion gel (union). This fusion gel was used for spot detection and spot mask editing. The final spot mask was transferred to each gel of the project to ensure 100% spot matching. The spot volumes of all 952 protein spots obtained were normalized by using total normalization. The protein synthesis ratios of the mupirocin-stressed samples and the corresponding protein spot of the control sample were calculated. Since we observed a general strong inhibition of the synthesis of proteins involved in the translation, we did not use an absolute threshold. Instead, we determined the 10% of the proteins with the highest induction ratios and the 10% of the proteins with the lowest induction ratios for each single sample point. Protein identification was performed as described previously (117) or deduced from the 2D protein reference map of S. aureus COL published elsewhere (9).

RNA preparation.

Total RNA was isolated from S. aureus COL using the acid-phenol method (48, 82) with modifications described by Fuchs et al. (44). The RNA for the DNA microarray experiments was further purified in order to eliminate traces of contaminating DNA. Briefly, the RNA was incubated with 7 U RNase-free DNase (Qiagen) for 10 min at room temperature, followed by two phenol-chloroform-isoamyl alcohol and two chloroform-isoamyl alcohol extraction steps in order to remove the DNase. The integrity of the RNA was checked with a Bioanalyzer (Agilent Technologies, Palo Alto, CA), and the concentration and purity were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Rockland, DE).

Northern blot analyses.

Samples for the Northern blot analyses were harvested at 5, 10, 30, and 60 min after mupirocin treatment. As controls, two samples were removed from the untreated culture at time point zero and 60 min after the beginning of the stress experiment. Northern blot analyses were done according to the protocol published by Wetzstein et al. (116). Digoxigenin-labeled RNA probes were prepared by in vitro transcription with T7 RNA polymerase and appropriate PCR fragments as templates (123). The PCR fragments were generated with chromosomal DNA isolated from S. aureus COL using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) and the oligonucleotides listed in Table 1. Digoxigenin-labeled RNA marker I (Roche, Indianapolis, IN) was used to estimate transcript sizes. The hybridization signals were detected using a Lumi-Imager (Roche Diagnostics, Mannheim, Germany) and analyzed using the software package Lumi-Analyst (Roche Diagnostics, Mannheim, Germany).

Table 1
Oligonucleotides used in this study

DNA microarray analyses.

Samples for DNA microarray analyses were harvested at time point zero and 10 min after the addition of mupirocin.

Synthesis of Cy5-dCTP- or Cy3-dCTP (PerkinElmer, Waltham, MA)-labeled cDNA was done with 10 μg of total RNA as a template by direct reverse transcription using Superscript II (Invitrogen, Darmstadt, Germany) and random hexamers (Promega, Madison, WI) according to the manufacturers' instructions. To degrade the RNA after cDNA synthesis, the reaction mixture was incubated for 30 min at room temperature with E. coli RNase H (Invitrogen, Darmstadt, Germany). The labeled cDNA was then purified with the QIAquick PCR Purification Kit (Qiagen, Mannheim, Germany). The Cydye incorporation was analyzed with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Rockland, DE). A sample volume of the labeled cDNA corresponding to 30 pmol incorporated dye was used for two-color competitive hybridization experiments. In total, four independent hybridization experiments, with each representing a biological replicate including a control and a treated sample, were carried out. To account for the dye bias, two of the four replicates were dye swapped.

The design and evaluation of the customized StaphChip oligoarray manufactured by Agilent Technologies (Palo Alto, CA) used in this study have been described previously (24). The StaphChip used in the study is based on the whole genome sequences of S. aureus strains COL, MRSA252, MSSA476, Mu50, MW2, N315, USA300, and 8325. Hybridization and washing of slides were carried out as described by Charbonnier et al. (24). The slides were scanned with the Agilent scanner. Spot intensity values were extracted with the Feature Extraction software provided by Agilent. Visualization and analyses of expression data were done in GeneSpring GX 7.3.1 (Agilent) and with the Cyber-T program for the analysis of paired expression data (http://cybert.microarray.ics.uci.edu) (7). The parameters for the Bayesian standard deviation estimation applied in the Cyber-T analysis were as follows: a sliding-window size of 101 and a confidence value for the Bayesian variance estimate of 12. Genes with a P value of <0.001 associated with the Bayesian t statistic and at least a 2-fold change in the mean fluorescence value of the four hybridization experiments were considered biologically significant expression changes.

Metabolome analysis.

S. aureus wild-type strain COL was grown as described above in defined synthetic medium without addition of MOPS (morpholinepropanesulfonic acid) buffer. At an OD500 of 0.5, the cells were treated with 0.03 μg/ml mupirocin, and samples were harvested directly after the addition of mupirocin (0 min) and after 10 min and 30 min. Samples for intracellular metabolite analysis were taken by fast filtration through a 0.45-μm sterility filter by vacuum. The cells were washed and quenched, and metabolites were extracted as described by Meyer et al. (85). Analytical biochemistry detection of nucleotides was performed by ion-pairing–liquid chromatography–mass spectrometry (IP-LC-MS) (79) and detection of amino acids, organic acids, and fatty acids by gas chromatography-mass spectrometry (GC-MS) (78). Quantification of compounds was done by referring to an internal standard in all samples, Br-ATP for IP-LC-MS and ribitol for GC-MS analysis. Identifications and calibration curves for all metabolites were done through comparison and measuring of pure standard compounds; an exception was (p)ppGpp. For the latter, no chemical standard was commercially available, so the accurate mass was calculated and compared with the found mass [(M-H+)calculated = 681.911 versus (M-H+)detected = 681.910]. Quantification of (p)ppGpp was done by adopting the calibration values from ppGpp, due to the analogous structure. Extracellular-metabolite quantifications of sterile filtered culture medium were made by 1H nuclear magnetic resonance (NMR) analysis (78). The results for intracellular metabolites were normalized to the cell dry weight.

Microarray data accession number.

Transcription data were deposited at the NCBI Gene Expression Omnibus (GEO) database with accession number GSE30743 and platform identification GPL13937.


Extensive influence of mupirocin on global gene expression in S. aureus.

We used a combined functional-genomics approach, including proteomic, transcriptomic, and metabolomic analyses, to characterize the response of S. aureus COL to subinhibitory concentrations of mupirocin (Fig. 1).

Fig 1
Growth inhibition of S. aureus COL induced by mupirocin. S. aureus COL was grown in synthetic medium at 37°C and 134 rpm. At an OD500 of 0.5, the culture was split and treated with increasing amounts of mupirocin: 0 μg/ml (control), 0.015 ...

The analytical window of the 2D gel-based proteomic approach covered 679 identified individual protein spots. Since proteins may occur as multiple spots due to modifications changing their isoelectric points and/or molecular weights, the 679 identified spots were assigned to 516 individual proteins (D. Zühlke, S. Fuchs, H. Kusch, D. Albrecht, and S. Engelmann, unpublished data). Of these, 60 proteins (66 protein spots) showed decreased and 20 proteins (25 protein spots) increased synthesis in response to mupirocin treatment (Tables 2 and and33 and Fig. 2). The DNA microarray analyses identified 274 downregulated and 320 upregulated genes (see Tables S1 and S2 in the supplemental material). If we consider only transcripts for which a corresponding cytosolic protein spot was identified in the analytical window of our 2D gel (pI range, 4 to 7; mass range, 10 to 140 kDa), solely for 20% of the genes with altered transcript levels in response to mupirocin, similar changes in translational activity have been observed. On the other hand, for 40% of the differently synthesized cytosolic proteins, the observed effects on protein synthesis have been attributed to changed mRNA levels. These results clearly show that posttranscriptional regulatory mechanisms play an important role under these conditions, and only a combination of both techniques provides a comprehensive picture of the regulatory processes that effectively alter gene expression by mupirocin. A compilation of both methods identified a total of 318 negatively and 325 positively regulated genes, corresponding to 23% of the S. aureus COL genome coding capacity.

Table 2
Proteins with decreased synthesis after mupirocin treatment
Table 3
Proteins with increased synthesis after mupirocin treatment
Fig 2
Synthesis of cytoplasmic proteins following mupirocin treatment. (A) False-color image of 2D gels from l-[35S]methionine-labeled protein extracts. Green, untreated control (exponentially growing cells); red, cells exposed to 0.03 μg/ml mupirocin. ...

Among the repressed genes identified by both approaches, the most prominent functional groups were those with roles in the regulation, structure, and function of the cell's replication, transcription, and translation machineries. However, whereas the DNA array showed strong repression of 29 ribosomal protein-encoding genes, they were not detected satisfactorily by the 2D gels. Ribosomal proteins are characterized by an alkaline pI (usually in the range from 8 to 11) and are not reproducibly covered by the analytical window of the 2D gels used in this study (pI range, 4 to 7). We further observed strong repression of the pathways that provide precursors for RNA and DNA synthesis (the pur and pyr operons) (Fig. 3). This massive repression of genes whose products are required at a high level during rapid growth is a hallmark of the stringent response. Consistent with this, we observed that 10 min after the addition of mupirocin, the protein synthesis rate, as measured by the incorporation of radioactive methionine, had already decreased to 50% compared to that of the untreated control. Maximal repression of protein synthesis to 26% was observed 30 min after stress (Fig. 4).

Fig 3
Timeline analyses of newly synthesized proteins following mupirocin treatment. Shown are false-color images of the protein synthesis rates of S. aureus COL grown in synthetic medium under nonstressed conditions (exponential growth) (c1) compared to the ...
Fig 4
Inhibitory effect of mupirocin on the protein synthesis rate of S. aureus COL cells. Cultures were grown in synthetic medium and treated with 0.03 μg/ml mupirocin at an OD500 of 0.5. Five, 10, 30, and 60 min after the addition of mupirocin, the ...

Additional metabolic pathways that were downregulated included fatty acid and phospholipid biosynthesis and the tricarboxylic acid cycle. Furthermore, expression of genes of the phosphotransferase system (PTS) was repressed. In addition, the synthesis of several transport systems was repressed. Most of these transport systems encode highly hydrophobic integral membrane proteins and were mainly detected by the DNA microarray experiment. However, in some instances, the ATP-binding protein domains of ABC transporters (e.g., SACOL1108) were also detected as repressed on the 2D gels.

The resources released by the general shutdown of energy-consuming processes required during rapid growth are a prerequisite for the initiation of adaptation strategies in order to restore conditions that allow survival under stringent control. By far the strongest induction of expression was observed for the isoleucyl-tRNA synthetase IleS, the molecular target of mupirocin, and for the enzymes catalyzing branched-chain amino acid (BCAA) biosynthesis (the ilv-leu operon). Nonetheless, the intracellular levels of the BCAAs valine, leucine, and isoleucine increased only slightly 30 min after mupirocin treatment compared to the untreated control (Fig. 5). This lack of increased BCAA synthesis, albeit strong induction of the biosynthetic pathways and putative uptake systems, might be caused by allosteric inhibition of the biosynthetic enzymes by the isoleucine that is still present (41, 58, 106). Surprisingly, overall we observed only minor alterations in the intracellular amino acid and carbon metabolism intermediate pool and no specific changes in the extracellular metabolite concentration following mupirocin treatment. No significant changes in the concentrations of glycine, tryptophan, threonine, aspartate, glucose, pyruvate, phosphoenolpyruvate, citrate, fumarate, ketoglutarate, malate, glycerol, lactate, and urea were detected. The succinate concentration displayed a significant drop at 30 min (data not shown), and the nucleotide pools changed in distinct ways in the mupirocin-treated culture.

Fig 5
Effects of mupirocin on Ala, Leu, Val, Ile, and Thr amino acid biosynthetic pathways. (A) Schematic overview of biosynthetic amino acid pathways summarizing the results of the proteome and the array analyses. Significantly induced or repressed synthesis ...

In addition to ileS and the ilv-leu genes, several genes with roles in adaptation to stress and starvation were significantly induced (ahpC, katA, SACOL1753, SACOL1759, and SACOL2131). Of particular interest was the induction of the two GTP pyrophosphokinases, encoded by relA1 (synonyms relQ and SACOL1010) and relA2 (synonyms rsh and SACOL1689), responsible for the synthesis of the stringent response alarmone (p)ppGpp (for a review, see reference 118). Furthermore, toxin/antitoxin addiction modules, encoded by SACOL2404-SACOL2405 and SACOL2464-SACOL2465, were among the induced genes. In addition, Northern blot analysis revealed upregulation of the transcription of the genes SACOL2059-SACOL2058, coding for MazE-MazF, which are not represented on the DNA array. Another striking observation was the induction of a large set of operons encoding ABC transporters and permeases with a predicted substrate specificity for amino acids and oligopeptides. Regarding the above-mentioned results, it is important to note that almost 50% of the genes that we found to be either induced or repressed in response to mupirocin encode hypothetical proteins or proteins with unknown or only predicted functions.

While the DNA microarray analyses reflected the immediate events in the adaptation to a mupirocin challenge, the proteomic data allowed timeline analysis. This analysis revealed two groups of expression kinetics for the induced proteins: (i) proteins with an immediate increase in synthesis (e.g., IleS, enzymes of the BCAA pathway, and SACOL1759) already reaching the maximum induction level 10 min following mupirocin treatment and (ii) proteins with delayed upregulation of synthesis (e.g., SACOL2131 and AhpC) (Fig. 3 and Fig. 5). Expression of proteins in the second group increased at a constant rate during the course of the experiment. These results suggest that the disturbance of the cells' physiology caused by mupirocin, in addition to stringent conditions, evokes secondary stress that may accumulate over time.

A clear grouping of significantly repressed proteins was less evident. In the majority of cases, repression was already observed 10 min after stress, with maximum repression occurring between 30 and 60 min following mupirocin exposure (Fig. 3). In general, the translation kinetics equaled the transcription kinetics, as shown for selected induced and repressed genes (typA, codY, ileS, ilvD, SACOL1759, and SACOL2131) by Northern blots (Tables 2 and and33 and Fig. 6).

Fig 6Fig 6Fig 6
Northern blot analysis of genes whose transcription was influenced by mupirocin. For RNA preparation, S. aureus COL was grown in synthetic medium at 37°C to an OD500 of 0.5 and treated with 0.03 μg/ml mupirocin. RNA was isolated 5, 10, ...

For five genes (cap5G, serA, SACOL2136, SACOL2293, and SACOL2597), however, an opposite regulation was observed in the 2D gel and DNA microarray experiments. For instance, while the DNA array detected clear induction of SACOL2597 transcription, the proteomic approach revealed strong downregulation in synthesis of the encoded protein. In this context, it is important to consider that the signal in the DNA microarray reflects the sum of all RNA molecules detected by a given probe. For SACOL2597, Northern blot analyses showed a complex transcript pattern. The small size of some of the detected mRNAs suggests processing of the SACOL2597 mRNA and may thus point to posttranscriptional regulation of SACOL2597 expression via control of mRNA stability (Fig. 6).

Regulatory networks active in the presence of mupirocin. (i) (p)ppGpp.

The massive changes in gene expression following mupirocin treatment are orchestrated by an exceedingly complex network of global regulators that act at the transcriptional and posttranscriptional levels. The major player in this network is likely (p)ppGpp, and intracellular accumulation of (p)ppGpp was evident 30 min after mupirocin treatment (Fig. 7) (22, 29, 46, 51). The amount of pppGpp exceeded that of ppGpp, which is in agreement with previous data (22, 46). A significant decrease in the level of the (p)ppGpp reagent GTP was already measurable 10 min after the addition of mupirocin. At this early time point, (p)ppGpp was not detectable. These data suggest that the significant drop in the GTP level may exert a major regulatory effect, since considerable alterations in gene expression were already observed 10 min after stress. At present, however, the possibility that biologically effective levels of (p)ppGpp may have escaped detection by the liquid chromatography (LC)-based approach cannot be excluded, since transcription of the (p)ppGpp synthetase gene relA1 (relQ) and the bifunctional (p)ppGpp synthetase and hydrolase gene relA2 (rsh) was already significantly upregulated at the 10-min point. In contrast, expression of SACOL2518, which is a homologue of the streptococcal (p)ppGpp synthetase gene relP, was downregulated. The exact physiological roles of RelQ and RelP in firmicutes has not been conclusively clarified yet, but it has been suggested that they may contribute to the fine tuning of the (p)ppGpp level under specific conditions. The main source of (p)ppGpp following amino acid limitation in S. aureus or Streptococcus mutans, however, remains synthesis by RelA2 (synonyms Rsh and SACOL1689) (46, 76, 118).

Fig 7
Intracellular concentrations of nucleotides involved in (p)ppGpp synthesis following mupirocin treatment. S. aureus COL was cultivated in synthetic medium to an OD500 of 0.5 and treated with mupirocin at a final concentration of 0.03 μg/ml. Shown ...

(ii) T-box.

In addition to the stimulation of (p)ppGpp synthesis, monitoring of the cellular amino acid pool via tRNAs is further exploited by a conserved cis-regulatory sequence motif, the T-box. A T-box is present in the leader region of many mRNAs encoding aminoacyl-tRNA synthetases and biosynthesis genes. Binding of the cognate uncharged tRNA to the T-box stimulates read-through of a termination signal in the mRNA leader region, allowing expression of the genes located downstream (53). The presence of a T-box has been demonstrated for the ileS leader mRNA (52). Inspection of the upstream region of SACOL2412, encoding an amino acid binding protein of an ABC transporter, which was strongly induced by mupirocin, also revealed the presence of a T-box motif. Thus, it is conceivable that expression of this gene, like that of ileS, is also subjected to control by isoleucyl-tRNA. In addition, the biosynthetic pathway for the synthesis of BCAAs encoded by genes of the ilv-leu operon was markedly upregulated at the transcriptional and the protein synthesis levels. Although only a weak T-box motif was identified in the ilv-leu upstream region, isoleucyl-tRNA-dependent regulation of the ilv-leu operon cannot yet be fully excluded.

In contrast to the strong upregulation of ileS, the alanyl-tRNA synthetase (alaS), histidyl-tRNA synthetase (hisS), and leucyl-tRNA synthetase (leuS) genes were downregulated. This might be a result of the accumulation of their cognate charged tRNAs due to the overall stop in protein synthesis, since good T-box sequences were identified in the upstream regions of the respective genes.

(iii) CodY.

Although it is obvious that mupirocin triggers a stringent response, in the absence of a mutant defective in synthesis of (p)ppGpp, we cannot clearly distinguish between regulatory effects directly [(p)ppGpp mediated] or indirectly related to stringent control. The large number of regulated genes suggests that, in addition to stringent control, further global regulators, like CodY, shape the response of S. aureus to mupirocin (46).

CodY is a global transcriptional repressor in many Gram-positive bacteria that during exponential growth mainly inhibits transcription of a large number of genes required for stationary-phase adaptation (10, 101, 107, 108). Although the amino acid sequence of CodY is highly conserved among firmicutes, CodY activity is differently affected by GTP and BCAAs. While in B. subtilis and its close relatives Bacillus cereus, Clostridium difficile, and Listeria monocytogenes CodY is allosterically activated for DNA binding by both GTP and BCAAs (11, 17, 33, 65, 87, 101, 105), in lactococci and streptococci, CodY activity seems to be enhanced solely by BCAAs (54, 61, 77, 97).

A consensus directed search using the B. subtilis CodY operator sequence AATTTTCWGAAAATT (31) as a query identified 63 genes potentially under the control of CodY in S. aureus COL. Of these, 19 were found to be upregulated and 4 to be repressed in our experiment. A significant overlap was also found with the experimentally determined CodY regulons in S. aureus Newman and UAMS-1. Of the genes affected by mupirocin in the present study, CodY has been shown to regulate the transcription of 44, and of 61 genes in strains Newman and UAMS-1, respectively. This corresponds to 35% (Newman) and 22% (UAMS-1) of CodY-regulated genes in these strains (80, 98).

Furthermore, the metabolome analyses for the two CodY ligands showed a marked decrease in the intracellular GTP pool but only a slight increase in intracellular BCAA levels (Fig. 5 and and7).7). Interestingly, following mupirocin treatment, we found a marked repression of CodY expression at the transcriptional and translational levels. However, autoregulation of the codY operon has not been demonstrated for S. aureus so far. Together, these observations suggest that CodY is a central regulator in S. aureus during the response to mupirocin and that in the absence of changes in the level of BCAAs a drop in the cellular GTP level may be sufficient to alter the affinity of CodY for at least some of its operators in S. aureus.

(iv) SigB.

The alternative sigma factor SigB (RpoF) may be an additional global regulator active in mupirocin-treated cells. Of the 25 genes with an experimentally confirmed SigB-dependent transcriptional start site (14, 32, 48, 62, 63, 83, 95), 16 showed increased expression following mupirocin stress in the present study (katA, opuD2, SACOL0444, SACOL0671, SACOL0678, SACOL0679, SACOL0680, SACOL0681, SACOL0682, SACOL0684, SACOL0685, SACOL0741, SACOL0742, SACOL2136, SACOL2174, and sarA) (Table 3; see Table S2 in the supplemental material). Interestingly, recent work showed that alkaline stress, a condition that was demonstrated to induce SigB-dependent transcription, also triggers a stringent response in S. aureus (4, 95). For a selected set of SigB-dependent transcripts (SACOL0742, SACOL2484, sarA, sigB, and spoVG), induction upon mupirocin treatment was confirmed by Northern blotting. These analyses further revealed a steady increase in the transcription of these genes during the course of the experiment, suggesting continuous SigB activation (Fig. 6). Surprisingly, transcription of the sigB operon, which is under autoregulation by SigB, showed transient repression immediately after mupirocin stress. In addition to increased SigB activity, we observed strong repression of the rpoD gene encoding the housekeeping sigma factor SigA. A model for passive sigma factor regulation was proposed for E. coli under stringent conditions (70, 74). Core RNA polymerase that is no longer required for rRNA synthesis may become increasingly available for interaction with alternative sigma factors, thus increasing transcription at the respective target promoters. A similar mechanism may also account for increased SigB activity under stringent conditions in B. subtilis (39). However, at present, it remains to be elucidated if such passive regulation of sigma factor activity also applies to SigB in S. aureus or if an as yet undiscovered signal regulates SigB activity in S. aureus during mupirocin treatment.

(v) Toxin-antitoxin systems.

Furthermore, it has been proposed that chromosomally encoded toxin-antitoxin systems might be integrated into the regulatory networks that are important in coping with stress by downregulating vital cellular processes ranging from transcription and translation to replication (47, 86, 89, 96). Among the mupirocin-induced genes, we identified three toxin-antitoxin systems, SACOL2405-SACOL2404, SACOL2465-SACOL2464, and mazE-mazF (SACOL2059-SACOL2058, also termed pemI-pemK). Recent work demonstrated UACAU sequence-specific endoribonuclease activity for MazF (43, 122). For SACOL2404 and SACOL2464, encoding homologues of E. coli YoeB, a function in the inhibition of translation initiation was shown (121). It is tempting to speculate that the above-mentioned S. aureus toxin-antitoxin systems are active under stringent conditions and therefore contribute to the fitness of S. aureus following mupirocin treatment. Transcription of these systems was also shown to increase upon treatment with tetracycline and erythromycin (34) and thus may have an even more general function for survival in the presence of antibiotics that interfere with ribosome function.


A growing number of recent studies have established a close link between the stringent response and the capacity to cause disease for many pathogenic Gram-positive and Gram-negative bacteria. In general, the capacity to initiate a stringent response is crucial to sustain virulence. The precise link between the stringent response and virulence, however, seems to be unique for each pathogen (1, 18, 45, 49, 56, 69, 115). For S. aureus, it has been shown in different animal models that persistent activation (45), as well as permanent inhibition, of the stringent response (46) may not be beneficial and rather may lead to attenuated virulence. Consistent with this, we found a number of virulence factor genes (seb, fnbA, fnbB, epiG, epiE, SACOL1164, SACOL1169, sdrD, sdrC, SACOL2019, and efb) to be upregulated following a mupirocin challenge that might be stringently controlled. Of these, at least five have a function in adhesion of S. aureus to host tissue or serum proteins.

Moreover, induction of SACOL1891, encoding the RNAIII-activating protein TRAP, was almost 5-fold upregulated following mupirocin stress compared to the control conditions. Interestingly, the TRAP protein, in addition to the modulation of RNAIII activity, may have a function in oxidative-stress resistance (71). A particularly high level of induction of TRAP was also observed after diamide stress (117).

Furthermore, the staphylococcal enterotoxin B gene (seb) and the similarly upregulated SACOL2449, encoding a hypothetical drug transporter, possess putative CodY binding motifs. By lowering the stringency of the motif search, fnbA and the cap operon, also positively controlled, were identified as potential CodY target genes (98). In S. aureus, CodY was demonstrated to serve as a regulatory switch that matches the cellular metabolic state with the expression of virulence factors whose functions may be related to the recruitment of nutrients from the host environment (81, 98, 111).

For the more classical virulence regulators, we observed enhanced transcript levels for genes encoding members of the Sar family (sarA, sarS, and sarR) and of two-component systems (saeRS and arlRS). A recent study demonstrated that upregulation of sarA and sae mRNAs in the presence of mupirocin is independent of relA2 (rsh) but may be the consequence of transcript stabilization, as shown for sarA (46). The target genes of these virulence regulators are often coregulated by the cell density-dependent agr two-component system (26, 27, 102). Since the present study was done under condition where the agr system was not active, the effect of mupirocin on expression of agr-regulated genes cannot be conclusively addressed.

Genes with conserved induction under stringent conditions.

In order to identify key genes important for coping with stringent conditions, we compared our data with previous analyses done in S. aureus and B. subtilis (3, 40) (see Fig. S1 in the supplemental material). While the global repression of the energy-consuming cellular machineries required for growth and proliferation is a highly conserved feature of the stringent response, a large variation was observed for the expression of genes with functions in energy metabolism, as well as transport and binding processes. This may reflect differences between the stimulus inducing the stringent response and the growth media used in these studies. Interestingly, the largest variation was found for upregulated genes encoding hypothetical proteins (see Fig. S1 in the supplemental material), suggesting that many of them may have a role in the fine tuning of the cell's physiology in response to growth-limiting conditions.

Among the staphylococcal genes induced in our work and that of Anderson et al. (3), SACOL1753, SACOL1759, and SACOL2131 are of particular interest. The first two are members of the universal stress protein (Usp) family, which can be found in bacteria, archaea, and eukaryotes (73). In E. coli, the Usp proteins have multiple functions in adhesion, motility, stationary-phase survival, and oxidative-stress resistance (90, 94). For the M. tuberculosis Usp Rv2623 protein, a role in the establishment of chronic persistent infection was demonstrated (35). SACOL1759 expression increases under many additional stress conditions (44, 117) in S. aureus and thus may have a versatile function in stress resistance. In the case of SACOL1759, two distinct protein spots were identified, with an increased synthesis rate in the 2D gels. The second, more acidic spot (SACOL1759-2), especially, displayed a very high induction ratio at 30 min after mupirocin treatment, suggesting that the function of SACOL1759 is regulated at the posttranslational level by an as yet unidentified protein modification.

SACOL2131 (also known as MrgA) is a Dps-like protein. These proteins act as DNA-binding ferroxidases, thereby decreasing hydroxyl radical formation in the vicinity of DNA (55, 84). It has been demonstrated that SACOL2131 is under double control by PerR and Fur in S. aureus (88). Interestingly, we also observed increased expression of the PerR-regulated gene ahpC and the ferritin encoded by ftnA (SACOL1952) (64).

Extending our comparison to B. subtilis, besides the ilv-leu operon, SACOL0815 (yvyD), SACOL0413 (ydaF), and SACOL2379 (ydaG) were also induced in all three studies (the respective B. subtilis genes are shown in parentheses). SACOL0815 (yvyD) encodes a protein highly similar to the E. coli ribosome-associated translation inhibitor RaiA. RaiA decreases aminoacyl-tRNA affinity for the ribosomal A site following various physiological stresses to prevent miscoding and improve translation fidelity (2). In B. subtilis, yvyD is under double transcriptional control by the two alternative sigma factors SigB and SigH (36) and associates with the ribosome (J. Muntel and D. Becher, personal communication). SACOL0413 (ydaF) codes for a putative ribosomal protein-serine acetyltransferase. Acetylation of ribosomal protein L12 increases the stability of the stalk complex under conditions of stress in E. coli (50). Indeed, our DNA microarray experiment identified 11 predicted acetyltransferases (including 7 from the GNAT family) as regulated by mupirocin, suggesting that protein modification by acetylation may be an important regulatory strategy during adaptation to stringent conditions.

Finally, sequence analysis of SACOL2379 (ydaG) shows homology to the FMN-binding domain present in pyridoxamine 5′-phosphate oxidases. YdaG is one of the few proteins common to the SigB regulons of S. aureus and B. subtilis (95). The function of YdaG, however, remains elusive.

The identification of marker genes and proteins, specific for classes of antibiotics with well-defined modes of action, has proven to be a valuable approach for the elucidation of the molecular targets of so far uncharacterized antimicrobial compounds (8, 42). For example, it has been demonstrated that strong upregulation of the target aminoacyl-tRNA synthetases and the respective amino acid biosynthesis pathways is a common feature of aminoacyl-tRNA synthetase inhibitors (8, 13, 42). As a proof of principle, Freiberg and colleagues identified a phenylthiazolylurea derivative as a novel phenylalanyl-tRNA synthetase inhibitor using DNA microarray-based reference expression profiles generated for a set of 14 well-characterized antibiotics (42). Interestingly, in the model organism B. subtilis, the expression of SpoVG, YvyD, and Dps, which was also upregulated by mupirocin in our study, was positively correlated with the treatment of different aminoacyl-tRNA synthetase inhibitors or the artificial depletion of aminoacyl-tRNA synthetases using conditional mutants (8, 13). These observations further support our conclusion that these genes may have important functions in the process of adaptation to amino acid limitation and hence the stringent response.


In summary, mupirocin treatment elicits a complex and comprehensive response in S. aureus that shows a clear link to the stringent response. In the absence of a mutant defective in the synthesis of (p)ppGpp, the main effector of the stringent response, however, it is not possible to clearly distinguish between stringent regulated genes and genes regulated by additional mechanisms. Global regulators, such as CodY, SigB, and chromosomally encoded toxin/antitoxin modules, likely contribute to the observed response. In the presence of mupirocin, derepression of CodY-controlled genes may be triggered by a drop in the GTP level rather than in the pool of BCAAs. Moreover, comparison to similar studies showed that growth conditions have a significant impact in individually shaping the response of S. aureus to mupirocin. Conserved induction of genes in S. aureus and B. subtilis (e.g., SACOL0815 [yvyD], SACOL0413 [ydaF], and SACOL2379 [ydaG]) following stringent conditions may point to important roles in the adaptation process. Furthermore, mupirocin clearly alters virulence gene expression in S. aureus. Targeting these genes may increase sensitivity to mupirocin and ultimately improve therapy and control of S. aureus infections.

Supplementary Material

Supplemental material:


This work was supported by the DFG (SFB/Transregio 34, Forschergruppe 585, and Graduiertenkolleg GRK 840) and the BMBF (Competence Network PathoGenoMik and ERANET Pathogenomics Network, sncRNAomics project).

We thank Decodon GmbH for the Delta 2D 3.4. gel analysis software and Dirk Albrecht for protein identification by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS).


Published ahead of print 21 November 2011

Supplemental material for this article may be found at http://aac.asm.org/.


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