- Split View
-
Views
-
Cite
Cite
Dacheng Wang, Lu Yu, Hua Xiang, Junwen Fan, Liang He, Na Guo, Haihua Feng, Xuming Deng, Global transcriptional profiles of Staphylococcus aureus treated with berberine chloride, FEMS Microbiology Letters, Volume 279, Issue 2, February 2008, Pages 217–225, https://doi.org/10.1111/j.1574-6968.2007.01031.x
- Share Icon Share
Abstract
In this study, we show that berberine chloride (BBR) has antimicrobial activities against all 43 tested strains of Staphylococcus aureus, an important human and animal pathogen. However, the response mechanisms of S. aureus to BBR are still poorly understood. Affymetrix GeneChips were used to determine the global transcription of S. aureus triggered by treatment with subinhibitory concentrations of BBR. 468 genes were up-regulated and 262 genes were down-regulated upon exposure to BBR. There was elevated transcription of various transporter genes, including genes involved in multidrug resistance, members of the multidrug and toxin extrusion family, the ferrous iron transporter, the amino acid transporter, the Na+/H+ antiporter, and the potassium cation transporter. Measurements of active transport were used to demonstrate a phenotypic correlation between efflux transporter overexpression and inhibition of BBR uptake. Furthermore, BBR induced the expression of urease genes, sortase enzyme, and iron-regulated surface determinant genes, but repressed transcription of a gene encoding arylamine N-acetyltransferase activity (N315-SA2490). To our knowledge, this is the first analysis of a genome-wide transcription profile of S. aureus cells in response to BBR treatment. These results will pave the way to exploring the mechanisms of BBR against S. aureus.
Introduction
Staphylococcus aureus is an important pathogen both in hospitals and in the community, and may cause a number of syndromes in humans, including endocarditis, osteomyelitis, and septicemia (Archer, 1998). Staphylococcus aureus is also the predominant cause of intramammary infection in dairy cattle, sheep, and goats, contributing to considerable economic loss (Anderson, 1983). The widespread use of methicillin and other semi-synthetic penicillins in the late 1960s led to the emergence of a strain of methicillin-resistant S. aureus (MRSA) (Ayliffe, 1997). More than 60% of S. aureus isolates are now resistant to methicillin, and a number of strains have developed resistance to more than 20 other antimicrobial drugs (Paulsen et al., 1997). The glycopeptide vancomycin was considered the last safeguard antibiotic against MRSA; however, the emergence of vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) has recently limited its effectiveness. Therefore, new agents are needed for the treatment of S. aureus.
Berberine, one of the major extracts of Coptis chinensis franch, is a protoberberine alkaloid that possesses antimicrobial activity against gram-positive and gram-negative bacteria, as well as against other microorganisms (Amin et al., 1969; Iwasa et al., 1998). Berberine also exhibits antimalarial, antisecretory, anti-inflammatory, and anticancer activities with relatively low cytotoxicity to human cells (Chung et al., 1999). Berberine inhibits the growth of streptococci and appears to prevent streptococci from adhering to host cells (Sun et al., 1988a). Although berberine does not inhibit the growth of Escherichia coli, it has been shown to block the adhesion of E. coli to erythrocytes and epithelial cells through the selective suppression of the synthesis and assembly of fimbriae (Sun et al., 1988b). It has also been demonstrated that berberine inhibits N-acetyltransferase activity and gene expression in Salmonella typhi (Wu et al., 2005).
Transcriptional profiles generated by GeneChip analysis of bacteria can provide valuable information with which to investigate differential gene expression in response to antimicrobial agents (Qiu et al., 2006). Transcriptional profiles have been used to identify genes in S. aureus strain 8325-4 that were induced in response to the cell wall active antibiotics oxacillin, d-cycloserine, and bacitracin (Utaida et al., 2003). Similarly, GeneChips were used to examine the effects of vancomycin on gene expression in S. aureus (McAleese et al., 2006).
In the present study, we investigated the antimicrobial activity of berberine chloride (BBR) by testing the minimum inhibitory concentrations (MICs) against clinical S. aureus isolates and a standard strain of S. aureus. Using an Affymetrix GeneChip, we analysed the global transcriptional patterns of S. aureus in response to subinhibitory concentrations of BBR.
Materials and methods
Bacterial strains and reagents
Forty-two clinical S. aureus isolates were obtained from the First Hospital of Jilin University, each with different antibiograms against 12 antimicrobial agents (data not shown). The standard strain ATCC25923 was obtained from the China Medical Culture Collection Center (CMCC). Mueller–Hinton broth II (MHB II) and Mueller–Hinton agar (MHA) were purchased from BD Biosciences, Inc. (Sparks, MD). BBR was purchased from Sigma-Aldrich (St Louis, MO), and stock solutions at various concentrations were made in dimethyl sulfoxide (DMSO) (Sigma-Aldrich).
Antibiotic susceptibility test
The MICs of BBR against 43 S. aureus strains were determined in triplicate by broth microdilution or broth macrodilution using twofold serial dilutions in MHB II according to CLSI/NCCLS M100-S15 (CLSI, 2005). The MICs were defined as the lowest concentration at which no visible growth was observed. The minimum concentration of BBR that inhibited 90% of the isolates was defined as MIC90.
Growth curves
Staphylococcus aureus strain ATCC25923 was grown to an OD of 0.3 at 600 nm in MHB II, and was distributed as 100-mL volumes into five 500-mL Erlenmeyer flasks. BBR (dissolved in DMSO) was added to four of the cultures to obtain final concentrations of 1/4 × MIC (32 μg mL−1), 1/2 × MIC (64 μg mL−1), MIC, or 2 × MIC (256 μg mL−1), respectively. The final DMSO concentration for all conditions was 1% (v/v). The control culture included the addition of 1% DMSO alone. The cultures were incubated further, and cell growth was monitored spectrophotometrically (OD600 nm at 15-min intervals). Three-milliliter samples of each culture were collected immediately after the addition of BBR (t0), and after 15, 30, 45, 60, 75, 90, 105, 120, 180, and 240 min. In addition, the total number of viable bacteria was estimated by plating dilutions of the culture on MHA without antibiotic and counting the numbers of CFU after 24 h at 37 °C.
Treatment with BBR
Staphylococcus aureus strain ATCC25923 was grown overnight in 10 mL of MHB II, at 200 r.p.m. in a rotary shaker at 37 °C. Two 250-mL Erlenmeyer flasks containing 100 mL of MHB II were inoculated with an overnight culture with an initial OD600 of 0.05. The bacteria were grown at 37 °C at 200 r.p.m. to an OD600 of 0.3. Then, 500 μL of 12 800 μg mL−1 BBR stock solution was added to one culture (the experimental group), and mock solution was added to the control culture. The final concentration of BBR in the experimental culture was 1/2 × MIC (64 μg mL−1). The final concentration of DMSO in each culture was 1% (v/v), and such amounts of DMSO did not alter the pH of the medium. The experimental and control cultures were incubated for a further 45 min at 37 °C, and RNA isolation was performed at this time. Three independent experiments were carried out.
RNA isolation and cDNA labelling
Bacterial cells were immediately treated with RNA Protect Bacteria Reagent (QIAGEN Inc., Valencia, CA) to minimize RNA degradation before harvesting. Subsequently, cells were collected by centrifugation and kept at −80 °C. Extraction of RNA was carried out using an RNeasy Mini kit (QIAGEN) according to the manufacturer's instructions. Contaminating DNA was then digested with RNAse-free DNAse I (10 U/40 μg of total bacterial RNA) at 37 °C for 20 min. RNA was repurified with an RNeasy Mini column (QIAGEN). RNA quality was monitored by agarose gel electrophoresis, and RNA quantity was measured by UV spectrophotometry. cDNAs were synthesized and labelled.
GeneChip hybridization and analysis
The commercial GeneChip S. aureus Genome Array (Antisense) used here was provided by CapitalBio Corporation (http://www.capitalbio.com/en/index.asp, Beijing, China), a service provider authorized by Affymetrix Inc. (Santa Clara, CA). The GeneChip includes N315 (National Institute of Technology and Evaluation, Japan), Mu50 (National Institute of Technology and Evaluation, Japan), NCTC 8325 (OU, lab strain), and COL (TIGR). The array contains probe sets to over 3300 S. aureus ORFs. In addition, the array contains probes to study both forward and reverse orientation of over 4800 intergenic regions throughout the S. aureus genome. Labelled cDNAs from independent RNA preparations were hybridized to six separate GeneChips. A total of 1.5 μg of labelled material was hybridized to each GeneChip for 16 h at 45 °C. After hybridization, washing and staining of arrays was performed using the GeneChip® Fluidics Station 450 and scanning with the Affymetrix GeneChip Scanner 3000, according to the manufacturer's instructions for antisense prokaryotic arrays (Affymetrix, Inc.).
The images were processed with Microarray Analysis Suite 5.0 (Affymetrix). The raw data from array scans were normalized by median-centring genes for each array, and log-transformed. Expressed genes were identified using affymetrix genechip operating Software (gcos, ver.1.0), which uses statistical criteria to generate a ‘present’ or ‘absent’ condition for genes represented by each probe set on the array. Subsequently, genes with ‘absent’ scores were filtered out and the remaining genes were analysed. significance analysis of microarrays (sam) software (http://www-stat.stanford.edu/~tibs/SAM/index.html. Tusher et al., 2001) was used to identify genes that are differentially expressed in BBR treatment samples compared with control samples. sam identifies genes with statistically significant changes in expression by assimilating a set of gene-specific t-tests, and provides an estimate of the false discovery rate (FDR) (the percentage of genes identified by chance alone) from randomly generated data. Genes with scores higher than a threshold value or genes with FDR values lower than the threshold value were deemed potentially significant.
In addition to microarray analysis, fold-change analysis was performed. Fold-change analysis included calculating the ratios of geometric means of expression intensities of BBR-treated samples, relative to controls. These ratios are reported as the up- or down-fold change. To select the differentially expressed genes, we used threshold values of ≥2 and ≤–2-fold change between BBR-treated samples and control samples and a FDR significance level of <5%.
Quantitative real-time reverse transcription (RT)-PCR
Quantitative real-time RT-PCR was used to verify the microarray result. Aliquots of the RNA preparations from BBR-treated and control samples used in the microarray experiments were saved for quantitative real-time RT-PCR. First-strand cDNAs were synthesized from 2 μg of total RNA in a 100-μL reaction volume using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Quantitative real-time PCR experiments were performed using the 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). All samples were analysed in triplicate and normalized against 16S rRNA gene expression. The cDNA was subjected to real-time RT-PCR using the primer pairs listed in Table 1. Cycling conditions were 48 °C for 30 min and 95 °C for 15 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min, and a dissociation step of 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s. Relative expression levels were determined by (ΔΔCt) method (Applied Biosystems User Bulletin no. 2).
ORF, open reading frame.
Refer to Luong (2006).
ORF, open reading frame.
Refer to Luong (2006).
Measurements of active transport
MIC analysis of BBR against ATCC25923 in the presence of reserpine (20 μg mL−1) was performed using broth microdilution (CLSI, 2005). The effect of reserpine (20 μg mL−1) on BBR uptake was also determined fluorometrically as described previously (Stermitz et al., 2000). Cells were cultured with aeration at 30 °C to an OD600 nm of 1.8, pelleted, and washed twice with 20 mM HEPES–NaOH (pH 7.0) buffer. The cells were then resuspended to an OD600 nm of 0.3 in 1 mL of HEPES buffer containing 10 μM glucose, followed by incubation at 37° or 30 °C for 1 h. The cells were centrifuged, washed, and resuspended at an OD600 nm of 0.15 in HEPES buffer. Assays were performed in 96-well flat-bottom black plates (Costar) in a final volume of 200 μL. At time zero, BBR was added at 30 μg mL−1 to the cell suspension to initiate the assay, reserpine was added at 20 μg mL−1 at the 9-min time-point, and fluorescence was measured at 3-min intervals with a Spectramax Geminis spectrofluorometer (Molecular Devices) at a 355-nm excitation wavelength and a 517-nm emission wavelength.
Results and discussion
Growth of S. aureus in the presence of subinhibitory concentrations of BBR
In this experiment, the MICs of BBR against 43 S. aureus strains ranged from 32 to 128 μg mL−1, and the MIC90 was 64 μg mL−1. The MIC of BBR against S. aureus strain ATCC25923 was 128 μg mL−1. The growth curve of S. aureus ATCC25923 is shown in Fig. 1. After 15 min of BBR treatment, there was no obvious difference in the OD600 nm value among all cultures. A steady increase in OD with 32 and 64 μg mL−1 of BBR treatment and the control occurred after 30 min. With 128 μg mL−1 of BBR treatment, the OD increased at a slower rate than it did with the lower concentrations. At the highest level of BBR, 256 μg mL−1, the OD600 nm increased initially and then decreased gradually. After 240 min, the OD600 nm value of the cultures treated with 32, 64, 128, and 256 μg mL−1 of BBR was c. 86%, 73%, 60%, 29% that of the control cultures, respectively. These results show that BBR concentrations of 128 and 256 μg mL−1 strongly inhibited the growth of S. aureus ATCC25923. In order to study the effects of a low berberine concentration on the transcription of the S. aureus strain, we reduced the inhibitory drug concentration to 1/2 × MIC (64 μg mL−1) according to the growth curve. Only the short-term (45 min) was examined in order to avoid confounding secondary drug effects. It has been claimed that lower concentrations are better for obtaining optimal microarray results, and that compounds should be at a low concentration in order to lessen the effect on the growth of the organism (Hutter et al., 2004).
Overview of transcriptional profiles
GeneChip analysis revealed that an enormous number of genes (730) were differentially regulated in response to BBR treatment. 468 genes showed a significant increase in transcription, and 262 genes showed a significant decrease in transcription. The microarray-related data were submitted to Gene Expression Omnibus (GEO) under accession number GSE7944. The distribution of BBR-responsive genes and their biological roles are shown in Fig. 2. A complete list of all genes differentially expressed by BBR treatment can be found in the supplementary material (supplementary Table S1).
In previous studies, the S. aureus Affymetrix GeneChips for Wyeth were used to analyse other antimicrobial agents such as oxacillin, bacitracin and d-cycloserine (Utaida et al., 2003), and vancomycin (McAleese et al., 2006). These S. aureus Affymetrix GeneChips have also been used to examine the effect of mild acid (Weinrick et al., 2004), cold shock, heat shock, and SOS response-inducing conditions (Anderson et al., 2006) on gene expression in S. aureus. The gene expression profiles showed components of general stress responses upon environmental challenges and stress-mediated changes in antimicrobial agent susceptibility. We compared the genes that were differentially regulated in the present study with those identified by the previous studies mentioned above. Herein, our interest was focused mainly on specific genes that may allow the organism to survive in the presence of BBR.
Up-regulation of transporter genes
Our results showed that a large number (98) of putative transporter genes of S. aureus were differentially regulated upon exposure to BBR (supplementary Table S1). Of these, some multidrug resistance (MDR) transporters were expressed at a relatively high level; for example, N315-SA0339 (3.5-fold increase), mepA (10.7-fold), N315-SA0263 (5.2-fold), N315-SA1982 (15.2-fold), N315-SA2241 (5.1-fold), and N315-SA1580 (3.1-fold). Interestingly, the mepRAB genes, associated with the multidrug and toxin extrusion (MATE) family, were up-regulated when S. aureus was exposed to BBR (supplementary Table S1). The MATE family of transporters has a broad substrate profile that includes several monovalent and divalent biocides and the fluoroquinolone antimicrobial agents. Furthermore, the three mepRAB genes are known to form an operon (Kaatz et al., 2005). We also found that BBR stress elevated the transcription of transport genes involved in the transport of ferrous iron (N315-SA2369, feoB), amino acids (N315-SA1270, braB, N315-SA2239), the Na+/H+ antiporter (N315-SA0582, N315-SA0583, N315-SA0581), and potassium cations (kdpABC).
In S. aureus, a major global regulator MgrA has been reported to affect several efflux pumps such as norA, norB, and tet38 (Truong-Bolduc et al., 2003, 2005). MgrA also appears to regulate a plethora of transporter proteins and transmembrane proteins, including sortase A (SrtA), preprotein transporter SecY, Na+/H+ antiporters, ABC transporters, amino acid transporters, ion transporters, a pyrimidine transporter, and sugar transporters (Luong et al., 2006). Among these MgrA-regulated genes, we found that N315-SA2239, N315-SA1674, N315-SA0531, N315-SA1270, N315-SA1675 were induced by BBR treatment, whereas N315- SA2311 was inhibited. These results are consistent with the regulation of mgrA. However, our results showed that mgrA down-regulated N315-SA0682 and up-regulated N315-SA1090, N315-SA1091, N315-SA1373, and N315-SA1374. These results are in contradiction to those from previous reports (Truong-Bolduc et al., 2003, 2005; Luong et al., 2006). It is likely that the discrepancy arises from the fact that the mgrA regulon was inhibited by a factor of 3.3 by BBR in the present study.
It is known that berberine is the substrate of the chromosomally encoded NorA S. aureus MDR pumps (Lewis, 2001). Surprisingly, the norA (N315-SA0650) gene was not induced nor inhibited by BBR in this study, the same result as described by other researchers (Kaatz & Seo, 2004). One explanation for this observation is that berberine does not interact with the presumed regulatory protein(s) and thus does not affect norA expression (Kaatz & Seo, 2004). Alternatively, berberine may increase norA transcription, but its action(s) on other cellular processes such as posttranscriptional regulation may indirectly result in reduced norA transcription. Thus, the observed gene expression is the result of the sum of stimulatory and repressive effects.
Berberine, an amphipathic cation, is the preferred substrate of most MDRs (Lewis, 2001). It was found that the MDR pump inhibitor 5′-methoxyhydnocarpin-d strongly increased the rate of penetration and the level of accumulation of berberine in S. aureus cells (Stermitz et al., 2000). In this study, measurements of active transport were performed to demonstrate a phenotypic correlation between efflux transporter overexpression and inhibition of BBR uptake. First, the effect of reserpine on BBR uptake was assayed using MIC testing. The results show that the MIC value for S. aureus ATCC25923 was reduced fourfold in the presence of reserpine, an efficient inhibitor of the MDR efflux pump inhibitor (EPI) (Gibbons & Udo, 2000), indicating that reserpine interacted with some efflux transporters to inhibit their efflux activity. Previous reports have shown that the efflux of cells overexpressing either MepA or NorA was significantly inhibited by reserpine compared with the respective untreated controls (Kaatz et al., 2000, 2005). In addition, berberine is a planar cationic molecule that resembles ethidium bromide and binds to DNA (Jennings & Ridler, 1983), and DNA-bound berberine has increased fluorescence. We took advantage of this property of berberine to directly examine the action of reserpine on BBR uptake using fluorescence techniques. The results of the fluorescence assay (Fig. 3) show that the addition of reserpine ultimately results in an increased BBR accumulation in S. aureus ATCC25923, consistent with the disruption of the function of one or more transporters.
Inhibition of the arylamine N-acetyltransferase gene
BBR repressed transcription of a gene encoding arylamine N-acetyltransferase (NAT) (SA2490) by a factor of 3.5. In previous studies, it was demonstrated that berberine shows dose-dependent inhibition of NAT activity, protein levels, and gene expression (NAT1 mRNA) in Salmonella typhi (Wu et al., 2005). It was also shown that berberine could markedly inhibit both NAT activity and the gene encoded for NAT1 in human brain tumour cells (G9T/VGH and GBM 8401) (Wang et al., 2002). Based on the decreased value of the kinetic constant of NAT, it was suggested that berberine might act as an uncompectitive inhibitor (Grant et al., 1991). Arylamine NAT is widely distributed in many species, including eukaryotes and prokaryotes. In this study, its activity was inhibited by berberine, consistent with the results from Salmonella typhi.
Induction of genes associated with sortase enzyme and iron-regulated surface determinants
In this study, the sortase-encoding gene srtB (SA0982) was induced by a factor of 2.7, and several surface protein-encoding genes isdCDEFGI were also induced in the response to BBR. In pathogenic gram-positive bacteria, the isd system (iron-regulated surface determinant) was found to encode factors responsible for haemoglobin binding and passage of heme-iron to the cytoplasm, where it acts as an essential nutrient (Skaar & Schneewind, 2004). Staphylococcus aureus Isd is composed of 10 genes, namely isdABCDEFGHI and srtB encoding cell wall-anchored haemoglobin, IsdB, hemoglobin–haptoglobin (IsdH/HarA) receptors, two cell wall-anchored heme binding proteins (IsdA, IsdC), a membrane transport system (IsdDEF), two cytoplasmic heme-degrading monooxygenases of the IsdG family (IsdG, IsdI), and a transpeptidase (SrtB) (Skaar et al., 2006). Pathogens such as S. aureus require iron to survive and have evolved specialized Isd proteins to acquire heme from their host (Skaar & Schneewind, 2004).
Increased transcripts of urease genes
The eight genes that constitute the ure operon (ureABCDEFG and the putative urease transporter SA2081) were up-regulated by BBR. Urease (urea amidohydrolase) is a nickel-containing enzyme that catalyzes the hydrolysis of urea to yield two molecules of ammonia and one molecule of CO2. Ureases of most bacteria are composed of three distinct subunits encoded by three contiguous genes, ureA, ureB, and ureC. Urease gene clusters also encode accessory genes, in addition to these structural genes, which are required for the de novo synthesis of active urease (Beenken et al., 2004). In Helicobacterpylori, urease is expressed at very high levels, and a shift to an acidic pH may result in a significant increase in the level of ure operon mRNA (Phadnis et al., 1996). In other bacteria, the urease activity is regulated in response to environmental changes, such as changes in pH, urea and nitrogen availability, and to growth phase (Burne & Chen, 2000). A recent report suggested that enhanced urease activity seemed to be an important acid-shock mechanism for S. aureus (Bore et al., 2007). The trigger for induced transcription of the urease operon in S. aureus by BBR challenge requires further study.
agr-Regulated genes affected by BBR
We found two-component system (TCS) genes agrAC were down-regulated by BBR. In S. aureus, virulence factors are coordinately regulated by a network of regulatory genes, which can be grouped into two major classes, TCSs and small transcription regulators. Among these TCSs, the accessory gene regulator (agr) system is the best-characterized system, and is composed of the AgrBDCA structural genes and RNAIII, the effector molecule of the agr locus (Novick, 2003). In our study, we found over 30 classical agr-regulated genes (Dunman et al., 2001) differentially expressed after BBR treatment (genes in boldface in supplementary Table S1). Strikingly, expression of most these genes was in the same direction (up- or down-regulation) as for the agr-regulated genes previously reported (Dunman et al., 2001), whereas agrAC were down-regulated by BBR treatment. It is likely that the discrepancy is a result of the inhibition of mgrA by BBR. It has been reported that MgrA is an activator of agr expression (Manna & Cheung, 2006). Moreover, we observed that an additional 11 TCS genes were also affected by BBR. Of these, vicR, glnA, phoR, phoP, SA0814 and SA0342 were down-regulated, and kdpABC, lytR and vraB were up-regulated. Thirty-five transcription regulator genes, including mgrA, were affected by BBR treatment. This suggests that the differential expression of virulence factors is coordinately regulated by these two-component signal transduction systems and transcription regulators.
Validation of microarray data by real-time RT-PCR
Real-time quantitative RT-PCR was conducted to validate microarray data using the same RNA as in the original microarray experiment. Eleven genes of interest were selected (mepA, kdpA, SA0339, vraB, ureB, spa, ebpS, glnR, agrA, SA2490, and isdA). In general, there was positive correlation between microarray data and real-time RT-PCR data for all 11 genes (Table 2), as five genes showed up-regulation and six genes showed down-regulation in response to BBR treatment. However, some genes (mepA, kdpA, ureB, glnR and SA2490) were changed to a greater degree in real-time RT-PCR than they were in microarray analysis. These results indicate that real-time RT-PCR analysis may be of greater dynamic range. In addition, poor or absent hybridization signals were generated for one of the tested samples, and n-fold induction values can be under- or overestimated in microarry analysis (Liu et al., 2005).
−, indicates reduction; +, indicates increase. SDs were calculated based on three independent experiments.
−, indicates reduction; +, indicates increase. SDs were calculated based on three independent experiments.
Conclusion
In summary, antibacterial activities and growth-curve experiments showed that BBR could significantly inhibit the growth of S. aureus. We determined the expression profiles of BBR-treated S. aureus cultures, and showed that the transcription of several major functional classes of genes was affected. Genes that were specifically affected by treatment with BBR could potentially be used as signature genes. These changes in gene expression can be viewed as an attempt by the organism to survive in the presence of this toxic substance. These findings may have important implications for understanding the responsive mechanisms of S. aureus to BBR.
Authors' contribution
L.Y. and D.W. contributed equally to this paper.
Acknowledgement
This work was supported by the National Nature Science Foundation of China (No. 30671586).
References
Supplementary material
This material is available as part of the online article from: (This link will take you to the article abstract.)
Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
Editor: Ross Fitzgerald