Global analysis of transcriptomic responses
To evaluate the impact of temperature up-shifts on the transcriptomic profile of S. aureus ISP794, we sorted all genes whose transcript levels were ≥ 2-fold upregulated or down-regulated by 10-min up-shifts from 37°C to 43°C or 48°C. The transcript levels of 93 (ca. 4%) of 2410 evaluated genes showed ≥ 2 fold changes at 43°C, among which 39 were down-regulated and 54 upregulated. More extensive changes were recorded at 48°C, since 532 (22%) transcript levels showed ≥ 2 fold changes, with 232 genes being down-regulated and 300 up-regulated. The distributions of the responding genes based on COG functional categories are shown on Additional file 1. Since several COG functional categories included a mixture of annotated and poorly functionally characterized genes (e.g. transcription regulators), we listed all poorly characterized genes in the general function prediction only category (see also Additional file 2).
To provide some indication of basal gene activities under control conditions, we also provided (Additional file 3, 4 and 2) semi-quantitative estimates of normalized signal intensities recorded at 37°C, which were subdivided into four categories (see Methods). Indeed, the highest-intensity signals (75th to 100th percentile) were well correlated with the most abundant transcript products of S. aureus predicted to be highly expressed from codon usage [34]. They also correlated quite well with the most abundant proteins revealed by S. aureus proteomic studies [35], in particular enzymes involved in DNA, RNA and protein transcription machineries, central metabolism and energy production. Conversely, the lowest intensity signals (25th percentile) recorded at 37°C were contributed by transcripts from poorly expressed genes, such as amino acid biosynthetic pathways known to be repressed by the presence of amino acids in the MHB medium [35].
Contribution of specific transcriptomic heat stress-responses
As expected from previous studies of heat-shock responses in gram-positive bacteria [13, 18, 19], all components of S. aureus HrcA and CtsR regulons [13] were strongly induced by up-shifts to both 43°C and 48°C (Additional file 3). Transcript levels of the genes regulated by CtsR only (ctsR, mcsA, mcsB, clpC, clpP, clpB) increased by ca. 3–5 fold at 43°C and ca. 3–11 fold at 48°C. We also observed increased expression of genes simultaneously regulated by HrcA and CtsR (grpE, dnaK, dnaJ, prmA, groEL, groES) at both 43°C and 48°C heat-shock. At 48°C, several HSP transcripts were detected at saturating levels by the microarray setting and thus their increased expression was likely under-estimated. To circumvent this problem and also validate the microarray-determined, heat-induced changes, we tested up-regulation of HSP transcript levels by qRT-PCR. Indeed, several gene transcripts (ctsR, mcsA, mcsB, hrcA) whose levels were saturated in the microarray scanner after up-shift to 48°C were more highly increased (ca. 6–16-fold) when assayed by qRT-PCR (Additional file 3). In contrast, after bacterial up-shift to 43°C, up-regulation of eight HSP genes (ctsR, mcsA, mcsB, clpC, clpP, hrcA, dnaK, and groEL) whose levels were not saturated in the microarray scanner, increased to a similar extent by qRT-PCR and microarray. These data confirmed the validity of microarray to quantify changes in bacterial transcript levels.
While the heat-induced upregulation of ctsR and hrCA may seem paradoxical in view of their previously described repressor activities [13, 18] that should down-regulate the transcription of other HSP genes belonging to their respective operons, other parameters may be involved to explain this paradox. First, it has been shown that the CtsR repressor needs ClpC protein to be active [18], and that high temperature may lead to accumulation of conformationally inactive CtsR in the absence of the chaperone co-factor [18]. Second, the global regulatory impact of ClpP protease on S. aureus virulence and stress responses also affects the regulation of genes of both the CtsR- and HrcA-controlled regulons [15]. Finally, significant heat shock-induced alterations in energy supplies, which may influence the availability of intracellular ATP levels required for Clp ATPases activities, might also have an impact on the transcriptional control of both CtsR- and HrcA operons.
Finally, to find out whether the presence of a fully functional SigB operon was required for heat-shock transcriptomic responses of HrcA- or/and CtsR-regulated HSP components, we also assayed by qRT-PCR the changes of HSP transcript levels in strain ISPU, a derivative of S. aureus strain ISP794 that was genetically restored with a complete rsbU+ operon. The 16-fold increase in transcript levels of the SigB-regulated gene asp23 confirmed RsbU restoration in the strongly pigmented strain ISPU compared to its non-pigmented RsbU-negative parent ISP794 (data not shown). Additional file 3 shows that heat-induced transcript levels in strain ISPU were either equivalent or <2-fold higher than those recorded in the RsbU-defective parental strain ISP794. Thus, a fully functional SigB operon was not required for induction of heat-shock regulons HrcA and CtsR.
In contrast to those heat-induced gene activities, serine protease HtrA-like (htrA) and trigger factor (tig) coding genes, as well as several other genes coding for Clp ATPases (clpL, clpQ, clpX, clpY) were not at all induced by up-shift to either 43°C or 48°C (Additional file 2), in agreement with previous observations [17, 18].
Finally genes coding for in situ repair mechanisms of damaged amino acid residues, such as those belonging to either the methionine sulfoxide reductase complex or the peptidyl-prolyl cis-trans isomerase protein PrsA [11, 36], were only marginally up-regulated by temperature up-shifts at 43°C or 48°C (Additional file 2).
Impact of heat stress on S. aureus growth and survival
Evaluation of S. aureus outcome following temperature up-shifts at 43°C or 48°C was performed by several assays. Both optical density measurements at OD540 and viable counts indicated that S. aureus cultures were in late-log phase during heat shock. In contrast to OD540 profiles that were not influenced by heat stress (data not shown), viable counts revealed that cultures up-shifted to 48°C did not grow further, and, if maintained at the same temperature, showed a slight, ca. 0.3 log10 decline in CFU/ml from 60 to 120 min after the heat shock onset. In contrast, CFU counts revealed that S. aureus cultures exposed to 43°C or 37°C showed equivalent growth and survival profiles. These data allowed defining 43°C and 48°C as sub-lethal and eventually lethal temperatures, respectively. We also observed that S. aureus cultures continually exposed to 43°C or 37°C showed marginally different growth kinetics, while those continuously exposed to 48°C remained growth-arrested at least for a 5 h-period (Figure 1) followed by a significant viability decline at 18 h (data not shown).
To verify that the marginal CFU decline during S. aureus heat stress at 48°C did not reflect heat-induced aggregation of the bacterial culture, we also evaluated bacterial viability by fluorescence microscopy, using the Live/Dead BacLight Bacterial Viability assay (see Methods). No significant aggregation was induced by heat exposure and the proportion of propidium iodide-stained, red bacteria increased slowly over time, in agreement with the slowly declining viable counts (data not shown). Finally, the extent of cell lysis was also estimated by the percentage of extracellularly released ATP before and after up-shift from 37°C to 48°C. The results showed nearly equivalent, low contents of extracellular ATP at the different temperatures, which represented <10% of intracellular ATP assayed in parallel and confirmed the marginal cell lysis (data not shown).
Additional heat-stress transcriptomic responses from various metabolic pathways
A large proportion of genes whose transcript levels showed ≥ 2-fold changes after up-shifts to either 43°C or 48°C belonged either to additional stress response pathways that were not regulated by CtsR-, HrcA-, and/or SigB, or to major metabolic pathways that likely contributed to the physiological adjustment and survival of heat-stress exposed bacteria. The Additional file 4 shows selected examples of up- or down-regulated genes representative of the different metabolic categories. A more exhaustive list of relevant gene transcripts and pathways is presented in the Additional file 2, which also includes altered genes of general function prediction only or unknown function.
Regulation of osmotic balance
Some heat-induced genes likely contributed to osmotolerance, such as those encoding glycine betaine transporter (opuD), choline dehydrogenase (betA), and glycine aldehyde dehydrogenase (gbsA). In contrast, transcript levels of the high affinity proline permease (putP) and proline betaine transporter (proP) remained constant, while those encoding the glycine betaine/carnitine/choline ABC transporter operon (opuCA/CB/CD), or the potassium-ABC transporter operon (kdpABC) and its regulator kdpDE, were down-regulated.
Oxidative stress responses
Some transcripts up-regulated by temperature up-shift at 48°C but not at 43°C were coding for enzymes coping with oxidative stress, in particular the superoxide dismutase gene sodA, and to a lesser extent (ratio: 1.84) thioredoxin (trxA) but not thioredoxin reductase (trxB). Occurrence of a heat-induced DNA damage at 48°C but not 43°C, potentially linked with oxidative stress, was suggested by increased transcript levels of nine genes coding for enzymes involved in DNA repair or/and recombination, namely dinB, uvrC, addA, recU, mutS2, the transcription-repair coupling factor mfd, the exonuclease SbcC, a zinc-dependent DNA glycosylase (SA1512), and to a lower extent polA encoding DNA polymerase I (ratio: 1.84). Part of those genes coding for DNA-damage repair and recombination enzymes were previously reported to be up-regulated, though to a variable extent, by S. aureus exposure to DNA-damaging agents such as mitomycin C [33] and ciprofloxacin [37], low pH [38], nitrite stress [39], peracetic acid [40] and cell-wall-active antibiotics [36]. In contrast, only one (uvrC) DNA-damage repair gene was up-regulated in S. aureus up-shifted to 43°C for 30 min [33]. In contrast to cell exposed to DNA-damaging agents [33, 37], we did not observe up-regulation of recA and lexA genes at 43°C or 48°C, which indicated the lack of a significant SOS response in heat-stressed bacteria.
Metal transporters
Several genes coding for influx or efflux metal transporters showed altered activities, which indicated possible dysregulation of metal homeostasis by temperature up-shifts. Except for the up-regulation of nixA coding for a high affinity nickel uptake transporter that seemed to be linked with urea cycle activation (see below), other up-regulated genes were encoding copper (copA) and zinc (czrAB) efflux transporters. Despite extensive studies, we lack a global, comprehensive model describing the regulation of physiological, intracellular levels of iron and other heavy metals in S. aureus, under normal and stressful conditions [41, 42]. While the peroxide operon regulator PerR was up-regulated at both 48°C and 43°C, transcript levels of some but not all PerR-regulated genes, such as katA (catalase), fnt (ferritin), and dps/mgrA also showed some increase at 48°C (see Additional file 2). The down-regulation of ABC transporter genes for other metallic cations such as manganese (mntABC) or cobalt might also indicate the need to avoid intracellular accumulation of potentially toxic levels of free heavy metals at 48°C.
Adjustment of ATP-providing pathways in heat-shocked S. aureus
Increasing, heat-triggered demand for protein- and DNA-repair mechanisms leads to higher consumption of cellular energy resources. Interestingly, nearly identical intracellular ATP levels were assayed in bacterial cultures shifted to 43°C (487 ± 7 pM/109 CFU), 48°C (483 ± 27 pM/109 CFU), or left at 37°C (469 ± 60 pM/109 CFU). Two previous reports also mentioned that heat stress did not decrease, but could even transiently increase, ATP levels in S. aureus [23] or E. coli [43]. To understand how heat-shocked bacteria could maintain constant intracellular ATP levels despite increased needs for repair systems, we evaluated gene expression changes in major energy-providing, metabolic pathways. Expression of genes encoding components of the glycolytic pathway remained quite constant after up-shifts to 43°C and 48°C, except for a nearly significant 2-fold decline of enolase (eno) at 48°C (see Additional file 2). More contrasting data were obtained with expression of TCA cycle genes, with three of them, namely citZ (citrate synthase), citC (isocitrate dehydrogenase), and odhB (dihydrolipoamide succinyltransferase), being up-regulated by heat-shock (48°C), while citB coding for the key TCA regulatory component aconitase was down-regulated [44]. It is unclear whether increased expression of citZ, citC, and odhB, which are conflicting with down-regulation of the TCA regulator aconitase, indicates an overall increased activity of the TCA cycle, or reflects individual contributions of some TCA components to other pathways. Indeed, citrate synthase may contribute to gluconeogenesis (by shuttling citrate to oxaloacetate and back to pyruvate/phosphoenolpyruvate) and dihydrolipoamide succinyltransferase to lysine degradation. Other microarray studies also reported induction of some TCA cycle components in stress-exposed S. aureus [37, 38]. Moreover, increased transcription at 48°C of zwf (glucose 6 phosphate dehydrogenase) and pycA (pyruvate carboxylase) also suggested activation of the pentose phosphate and gluconeogenesis pathways, respectively (Additional file 4).
We also noticed increased transcription at 48°C of three key enzymes (thiE, thiM, thiD) involved in the biosynthetic pathway leading to thiamine pyrophosphate coenzyme (ThPP), involved in major decarboxylation reactions of glycolysis, TCA and pentose phosphate pathways. A similar up-regulation of three key enzymes (ribA, ribB, ribD) coding for riboflavin synthesis was observed at 48°C. Both ThPP and FAD are also important for branched-chain fatty acid biosynthesis, derived from the catabolism of the branched-chain amino acids leucine, valine, and isoleucine [45, 46]. Moreover, increased expression of ThPP is also essential for biosynthesis of branched amino acids, and fit well with microarray data indicating derepression of 3 genes (leuA, leuB, leuC) coding for leucine biosynthesis. Adjustment of branched-chain fatty acid biosynthesis may be an important defense mechanism against heat-induced membrane disordering and contribute to restoring optimal membrane fluidity and proton impermeability [47] (see below).
Analysis of key metabolites in S. aureus growth medium
To evaluate the physiological significance of our transcriptomic data, we analyzed in parallel the presence of key metabolites in the growth medium, before and after temperature up-shifts. Freshly prepared MHB, before bacterial inoculation, contained relatively low levels of free glucose (0.38 mM), which were rapidly depleted (<0.001 mM) during the pre-shock growth period, as found in other studies [48, 49]. Extracellular starch levels, an abundant component of MHB, which was looked as a potential glucose-providing source, remained absolutely constant (assayed as 1.2–1.3 mg/ml of glucose equivalent) throughout bacterial growth. This suggested that S. aureus could not use starch as a nutrient source presumably because of the lack of extracellular amylolytic activity. Collectively, our transcriptomic and physiological data strongly indicated that, after glucose exhaustion from the medium, S. aureus was forced to use the most abundant alternative carbon sources that were amino acid or peptide mixtures provided in the casein acid hydrolysate component of MHB. Recent metabolic studies indicate that the catabolism of several amino acids can feed both TCA cycle and gluconeogenesis pathways by producing essential intermediates oxaloacetate, oxoglutarate, phosphoenolpyruvate, and pyruvate [44, 49, 50]. These metabolic studies also indicate that glucose depletion leads to derepression of TCA cycle components [44], as confirmed by our transcriptomic data showing their high expression levels at 37°C. While significant levels (3.0–3.5 mM) of acetate were detected in MHB just before and after temperature up-shifts, these levels remained marginal compared to those (ca. 15–20 mM) recorded in other studies [44, 48, 51], and were not sufficient to significantly acidify the growth medium. In contrast to gene activities of the glycolytic, pentose phosphate shunt, and TCA cycle pathways, most nitrate/nitrite reductase components were down-regulated at both 43°C and 48°C. Furthermore, several major fermentative pathway components were markedly down-regulated by heat stress at both 43°C and 48°C, in particular alcohol (adhE, adh1), lactate (ldhA, ldhB) and formate (fdh) dehydrogenases. Biochemical assays confirmed the marginal levels of L-lactate (0.3–0.5 mM) and D-lactate (< 0.15 mM) in MHB. The down-regulation of energy-providing fermentative pathways suggests that they may be energetically less efficient for heat-exposed S. aureus.
Adjustment of ATP-consuming pathways in heat-shocked S. aureus
Two categories of ATP-requiring biosynthetic pathways showed a significant, global reduction in transcript levels. The first category included the purine and pyrimidine synthetic pathways whose fifteen and nine components, respectively, were down-regulated to the same extent (Additional files 4 and 2). In contrast, transcript levels of drm (phosphopentomutase) and pnp (purine nucleoside phosphorylase), coding for salvage pathways, were markedly increased. Induction of salvage pathways contributes to the recycling of bases, by diminishing the high energetic costs of their multistep re-synthesis, and may also provide ribose to the pentose phosphate cycle. The second category of down-regulated transcript levels at 48°C included genes coding for 13 amino acyl-tRNA synthetases, among which eight were also decreased at 43°C (Additional files 4 and 2). Conversely, expression of cysteinyl-tRNA synthetase was significantly increased at 48°C.
In contrast, expression of most other genes coding for major biosynthetic apparatus of replication, transcription, and translation, e.g. ribosomal proteins, DNA or RNA synthesis, was not or only marginally affected by heat shock (see Additional file 2), except for rnc coding for RNase III whose expression was up-regulated at both 43°C and 48°C. A similar situation prevailed among cell wall and membrane biogenesis components, with only 10% of altered transcripts, in contrast to autolytic components whose expression was more affected by heat shock. Among cell division-regulating components, only scdA transcript levels, coding for a cell division and morphogenesis-related protein, were specifically reduced at both 43°C and 48°C.
Another category of ATP-consuming activities, whose expression appeared down-regulated, included 13 out of 15 evaluated ATP-dependent components of amino acid or peptide transporters (Additional files 4 and 2). Microarray data confirmed that amino acid/oligopeptide, transport was essential to cell metabolism because most amino acid synthetic pathways were repressed at 37°C. However, some of those amino acid pathways were strongly induced by up-shift to 48°C, as revealed by increased transcript levels (2.5–18 fold) of biosynthetic enzymes for lysine, tryptophan, glutamate, histidine, and branched chain amino acids. Up-regulation of those amino acid synthetic pathways, despite being high consumers of ATP, might indicate an increasing need of some amino acids during heat stress, possibly amplified by a decreased efficiency of some amino acid, ATP-driven transport systems. Of note, the content of free amino acids in MHB remained abundant throughout bacterial growth as well as after heat shock exposure (data not shown), which ruled out a specific depletion of some amino acids as observed in a previous study [49]. Therefore, the marginal decline in extracellular amino acid supply was not sufficient for explaining the selective, biosynthetic induction of some amino acids during heat stress at 48°C.
Since transcriptomic data suggest a decreased efficiency of energy-dependent transport systems in heat stressed-bacteria, this observation can be supported by the documented effects of increased temperature on bacterial membrane fluidity, which are known to alter proton impermeability and the proton-motive force [47, 52]. These heat-induced alterations in the membrane physico-chemical properties may require changes in its lipid composition for fluidity adjustment [47, 52].
Summary of transcriptomic and metabolic observations into a relevant metabolic model
Transcriptomic and metabolic data of S. aureus exposed to a sub-lethal (43°C) or eventually lethal (48°C) temperature can be summarized as follows: (i) heat stress exposure generates an increased ATP demand for protein- and DNA-repair; (ii) constant intracellular levels of ATP could be maintained despite a relative decline of ATP-generating sources, in particular fermentation and microaerophilic nitrate and nitrite reduction pathways. (iii) exhaustion of glucose supply during S. aureus culture preceding heat shock force the bacteria to feed ATP-generating pathways with amino acids metabolized into oxoglutarate, oxaloacetate, phosphoenolpyruvate and pyruvate, as essential TCA cycle and gluconeogenesis intermediates. We can further speculate that the decreased expression of a vast majority of amino acyl-tRNA synthetases might promote the release of amino acids that feed energy-providing pathways, though this may eventually compromise protein synthesis during prolonged heat shock.
The metabolic model proposed below (Figure 2) attempts to integrate metabolic responses (including already mentioned protein and DNA-repair pathways) of heat-stressed S. aureus with the predictable, heat-induced membrane disordering, in which increased motion of the lipid molecules may lead to increased proton transmembrane permeability and potentially severe bioenergetic consequences [47]. Studies in different bacterial species indicate that optimal membrane fluidity and proton impermeability can be restored by adjustment of its fatty acid composition [47, 52]. Major lipid biosynthetic pathways require high levels of NADPH and acetyl-CoA, which may explain up-regulation of the pentose phosphate cycle during heat shock. This may be further supported by up-regulation of ThPP and FAD biosynthetic pathways that are essential cofactors for biosynthesis of branched amino acids, whose catabolites are important precursors of branched-chain fatty acid biosynthesis [45, 46]. More detailed experimental studies are needed to confirm the importance of these adaptive mechanisms in S. aureus. Finally, the metabolic model also integrates the necessity for heat-stressed S. aureus to down-regulate the production of reactive oxygen species that may be generated via electron transport-generated ATP, in particular by reducing levels of free metals, such as iron, that may promote generation of superoxide and hydroxyl radicals [41, 42, 53].