ABSTRACT
σS is an alternative sigma factor, encoded by the rpoS gene, that redirects cellular transcription to a large family of genes in response to stressful environmental signals. This so-called σS general stress response is necessary for survival in many bacterial species and is controlled by a complex, multifactorial pathway that regulates σS levels transcriptionally, translationally, and posttranslationally in Escherichia coli. It was shown previously that the transcription factor DksA and its cofactor, ppGpp, are among the many factors governing σS synthesis, thus playing an important role in activation of the σS stress response. However, the mechanisms responsible for the effects of DksA and ppGpp have not been elucidated fully. We describe here how DksA and ppGpp directly activate the promoters for the anti-adaptor protein IraP and the small regulatory RNA DsrA, thereby indirectly influencing σS levels. In addition, based on effects of DksAN88I, a previously identified DksA variant with increased affinity for RNA polymerase (RNAP), we show that DksA can increase σS activity by another indirect mechanism. We propose that by reducing rRNA transcription, DksA and ppGpp increase the availability of core RNAP for binding to σS and also increase transcription from other promoters, including PdsrA and PiraP. By improving the translation and stabilization of σS, as well as the ability of other promoters to compete for RNAP, DksA and ppGpp contribute to the switch in the transcription program needed for stress adaptation.
IMPORTANCE Bacteria spend relatively little time in log phase outside the optimized environment found in a laboratory. They have evolved to make the most of alternating feast and famine conditions by seamlessly transitioning between rapid growth and stationary phase, a lower metabolic mode that is crucial for long-term survival. One of the key regulators of the switch in gene expression that characterizes stationary phase is the alternative sigma factor σS. Understanding the factors governing σS activity is central to unraveling the complexities of growth, adaptation to stress, and pathogenesis. Here, we describe three mechanisms by which the RNA polymerase binding factor DksA and the second messenger ppGpp regulate σS levels.
INTRODUCTION
In nature, bacteria are in constantly changing environments and are frequently assaulted by stressors. Accordingly, bacteria have evolved various cellular stress responses to survive and adapt to such unpredictable threats. One such adaptation is the ability to transition efficiently from exponential growth in rich nutrients to the stagnant, nutrient-exhausted state known as stationary phase. This transition into stationary phase is governed by the alternative sigma factor σS (RpoS) and is often referred to as the general stress response. Although σS induction is commonly associated with entry into stationary phase, it can also be triggered by several other environmental stresses, such as starvation for specific nutrients (e.g., carbon, nitrogen, or phosphate), oxidative stress, hyperosmotic shock, and changes in temperature or pH (reviewed in references 1 and 2). In response to these stressors, σS redirects RNA polymerase (RNAP) to transcribe a large family of genes known as the σS regulon that comprises approximately 10% of the Escherichia coli genome (3). These genes are responsible for a highly coordinated cellular response involving diverse functions, such as DNA repair, stress (ethanol, UV, or oxidation) protection, nutrient scavenging, and cellular maintenance (reviewed in reference 4).
σS has also been implicated in regulating the expression of virulence factors in some pathogens. For example, in Salmonella spp., σS is required for expression of both the plasmid-borne spv virulence genes and all five chromosomally encoded pathogenicity islands (5, 6). Further supporting the role of σS in virulence regulation, rpoS deletion mutants of Salmonella enterica serovar Typhimurium are attenuated in virulence and unable to cause infection in a murine model (7).
Because σS responds to multiple signals, the mechanisms governing its activity and expression are complex. trans-acting factors positively or negatively regulate virtually every step in σS gene expression, including rpoS transcription, mRNA transcript stability, translation of the rpoS mRNA, and σS protein stability. Transcription from the rpoS gene is initiated at an internal promoter within the upstream gene nlpD located 567 nucleotides upstream of the rpoS AUG start codon (8). This results in a long 5′ untranslated region (UTR) with a secondary structure that is the basis for posttranscriptional regulation of the rpoS transcript, because it creates an inhibitory stem-loop that obscures the ribosome binding site (9). It has been reported that the effects of this inhibitory stem-loop are alleviated by competitive binding of the DsrA (10, 11), RprA (12), and ArcZ small noncoding RNAs (13). These small RNAs (sRNAs) are assisted by the chaperone Hfq in binding to the 5′ UTR, revealing the translation start site and stabilizing the mRNA transcript (10, 14).
After translation and in the absence of stress signals, σS is recruited by the adaptor protein RssB, which delivers σS to the protease ClpXP for degradation (15, 16). Depending on the stress signal, the anti-adaptor protein IraP, IraD, or IraM binds and sequesters RssB, thereby preventing σS degradation and allowing its rapid accumulation (17). Together, this complex multifactorial regulation of σS levels allows an exquisitely fine-tuned cellular response for adaptation and survival in diverse, potentially harmful environments.
σS synthesis is also affected by the transcription factor DksA and the small signaling molecules guanosine tetraphosphate and pentaphosphate (abbreviated here as ppGpp) (18, 19). DksA is a transcription factor that binds in the secondary channel of RNAP, altering the kinetics of transcription initiation (20–22). As nutrients are depleted, ppGpp accumulates and, in conjunction with DksA, coordinates a global transcriptional response to alleviate the effects of the stress (23–25).
ppGpp and DksA directly and synergistically regulate large sets of promoters negatively (20) and positively (26). ppGpp binds to two sites on E. coli RNAP, site 1 (27), known to play a role in DNA break repair (28), and site 2 (29). The site 1 pocket is formed by the interaction of the β′ and ω subunits, and the site 2 binding pocket is formed by the interaction of DksA and the β′ rim helices. The requirement for both DksA and RNAP for formation of site 2 explains the DksA-ppGpp synergism (29). DksA and ppGpp are both required for full induction of σS in S. Typhimurium (30) and E. coli (19).
It was initially suggested that ppGpp regulates σS expression after transcription initiation, e.g., via effects on the rpoS mRNA (8), and subsequently, it was suggested that ppGpp helps induce σS at the translational level, likely in an indirect manner (19). Further clues resulted from studies on the anti-adaptor protein IraP and the small regulatory RNA DsrA. Cells deficient in ppGpp and DksA showed reduced expression of iraP, suggesting that the effects observed in vivo resulted from positive regulation of iraP transcription by DksA/ppGpp (31). A DsrA and ppGpp deficiency also reduced rpoS expression, but it was concluded that DsrA and ppGpp acted through independent pathways (18). DksA and ppGpp together also directly stimulated transcription from the promoter for Hfq, the protein that serves as a chaperone for most small RNAs (32), suggesting another potential contribution of DksA/ppGpp to rpoS expression. Finally, part of the activation of rpoS expression by DksA/ppGpp could occur through other indirect mechanisms. For example, DksA/ppGpp inhibition of rRNA transcription could lead to an increase in the availability of RNAP for utilization by promoters that otherwise recruit RNAP poorly (33) and/or inhibition of rRNA transcription could allow alternative sigma factors to compete better with the primary sigma factor for available core RNAP.
As a result of this complexity in rpoS regulation, the complete mechanism by which ppGpp and DksA contribute to the σS stress response remains unclear. To help clarify their roles in rpoS expression, here, we took advantage of DksAN88I, a variant with higher affinity for RNAP than wild-type (WT) DksA (34) that can enhance transcription of rpoS even in the absence of ppGpp in vivo. Our results provide support for a model in which DksA and ppGpp directly increase the expression of the DsrA small regulatory RNA and the anti-adaptor protein IraP, thus increasing σS levels. Furthermore, our results also suggest that DksA and ppGpp can increase dsrA and iraP expression indirectly by another mechanism, possibly by reducing rRNA transcription and thereby increasing RNAP availability.
RESULTS
DksAN88I activates the σS general stress response.To better understand the mechanism of DksA regulation of the σS stress response, we used the previously characterized variant DksAN88I, also referred to as super DksA (34). The N88I substitution is in the coiled-coil domain of DksA that inserts into the secondary channel of RNAP, increasing DksA's affinity for RNAP, thereby increasing its effects on transcription initiation. A key feature of this variant is that it can inhibit rRNA transcription dramatically compared to WT DksA and allow growth even in a strain lacking ppGpp in vivo (34). Thus, utilization of this super DksA allele provided a means of studying the effects of DksA in the absence of confounding effects of starvation or of ppGpp binding to cellular targets other than RNAP.
We first validated that DksAN88I, like WT DksA (29), activated the σS stress response in vivo. As an indicator of activation of a σS-dependent promoter and more broadly of the σS regulon in general (35), we examined expression of a PkatE-lacZ transcriptional fusion. Induction of plasmid-encoded DksAN88I strongly increased β-galactosidase (β-Gal) activity, and this increase was eliminated in a ΔrpoS mutant (Fig. 1A). Consistent with previous in vivo studies on PlivJ, a promoter activated by the stringent response (34), WT DksA overexpression was unable to induce PkatE in exponential phase (when ppGpp levels are very low) (Fig. 1A), whereas DksAN88I increased PkatE activity even in a ΔrelA ΔspoT (ppGpp0) background (Fig. 1B). As a result, DksAN88I induction resulted in accumulation of σS protein throughout exponential phase, allowing detection as early as an optical density at 600 nm (OD600) of 0.2 (Fig. 1C). In summary, these results show that DksAN88I strongly induces the σS stress response, as shown previously for WT DksA (19), and that DksAN88I can do so even in log phase.
Super DksA (DksAN88I) stimulates the σS general stress response and increases σS levels. (A) β-Gal activity was measured during exponential growth at 32°C in LB with 0.1 mM IPTG and was plotted against the OD600. Shown are PkatE-lacZ transcriptional fusions in WT (solid symbols) or ΔrpoS (open symbols) strains. Strains contained a plasmid expressing pDksAWT, pDksAN88I, or the pControl plasmid. (B) Same PkatE-lacZ transcriptional fusions as in panel A but in a ppGpp0 background (ΔrelA ΔspoT). Representative experiments are shown. (C) Western blot analysis of σS protein levels in WT cells with or without DksAN88I induction at OD600 values of 0.2, 0.4, 0.6, and 0.8 relative to pControl protein levels at an OD600 of 0.2. Bands from a representative Western blot are shown below the bar graph the error bars represent the means and SD from three independent experiments (n = 3).
Activation of the σS stress response by DksAN88I is not completely dependent on IraP in vivo.Under stress conditions, the posttranslational regulatory factor IraP prevents σS degradation by ClpXP protease, leading to accumulation of σS (17). Previously, it was reported that PiraP-lacZ activity was reduced in a ΔdksA strain (31), and this requirement for dksA for activation of PiraP was direct, since it occurred in vitro with purified DksA/ppGpp (29). Expression of DksAN88I, like that of WT DksA (29), activated a PiraP-lacZ fusion (Fig. 2A). To address whether the effect of DksAN88I on IraP accounted for the entire requirement for DksAN88I to increase σS levels, we measured the effect of DksAN88I on the PkatE-lacZ transcriptional fusion in a ΔiraP mutant (Fig. 2B). DksAN88I overexpression induced PkatE expression throughout exponential phase in a ΔiraP mutant, similar to the effect observed in a strain containing iraP (Fig. 1A and 2B). Thus, there must be a mechanism by which DksAN88I increases σS levels, in addition to its role in increasing expression of IraP.
DksA stimulates expression of IraP in vivo. Shown is β-Gal activity during exponential growth at 32°C in LB with 0.1 mM IPTG. (A) PiraP-lacZ transcriptional fusion with pControl or pDksAN88I in WT cells. (B) PkatE-lacZ transcriptional fusion in a ΔiraP mutant with pControl and DksAN88I. The β-Gal data are representative of at least three independent experiments. The absolute activities in panels A and B should not be compared directly, since the fusions are to different promoters transcribed by different holoenzymes.
DksAN88I specifically activates the expression of the small regulatory RNA DsrA in vivo.We next compared the effects of DksAN88I on σS levels in strains with two small RNAs previously implicated in regulation of σS, DsrA and RprA, deleted. Deletion of rprA only marginally reduced the effect of DksAN88I on σS levels, but deletion of dsrA (or both iraP and dsrA) virtually eliminated the effects of DksAN88I on σS levels (Fig. 3A). The effects of the double-deletion mutant were also examined at different times, from OD600 values of 0.2 to 0.8 (Fig. 3B). At each time point, the effects of DksAN88I were greatly diminished in the double mutant.
The effect of DksAN88I on σS levels is dependent on dsrA. Strains were grown to an OD600 of 0.6 in LB at 32°C with 0.1 mM IPTG. Shown is Western blot analysis of σS protein levels. (A) Protein extracts were made from the indicated strains containing pControl or pDksAN88I, and σS was normalized to the WT with pControl. A representative Western blot is shown below the graph. (B) Proteins were extracted from WT or ΔiraP ΔdsrA strains containing pControl or pDksAN88I as indicated. The bands were quantified and normalized relative to WT pControl (the error bars indicate means and SD; n = 3).
We next assessed the role of DsrA by measuring the effect of DksAN88I on an rpoS-lacZ translational fusion (36). There was a large increase in rpoS translation in exponential phase when DksAN88I was provided in trans, and this increase was largely eliminated in a ΔdsrA strain (Fig. 4A). Consistent with a role for factors in addition to dsrA in rpoS transcription, or even of posttranslational effects on the activity of the fusion protein, the residual activity of the translational fusion still increased slightly in the ΔdsrA pDksAN88I strain relative to the ΔdsrA pControl strain (Fig. 4A). However, the major conclusion was that DksAN88I greatly increased the expression of rpoS in a dsrA-dependent manner.
DksAN88I activates transcription of the small regulatory RNA DsrA in vivo. (A) β-Galactosidase activity from a PrpoS750-lacZ translational fusion in the WT (solid symbols) or a ΔdsrA mutant (open symbols) containing either pControl or pDksAN88I. (B) PdsrA-lacZ β-Gal activity plotted against increasing OD600 in WT background. (C) ParcZ-lacZ β-Gal activity plotted against increasing OD600 in WT background. The curves shown are representative of at least 3 independent experiments.
Since DksA (in conjunction with ppGpp) is a transcription activator, we tested whether it might exert its effect on rpoS expression by stimulating dsrA promoter activity in vivo. Consistent with this model, DksAN88I expression increased the activity of a PdsrA-lacZ promoter fusion (Fig. 4B). This result was also consistent with previous observations implicating DksA in expression of the small RNA chaperone Hfq, and thus of small RNAs, in regulation of rpoS synthesis by DksA (32). In contrast, DksAN88I expression did not increase the activity of a ParcZ-lacZ promoter fusion (Fig. 4C), suggesting that under the conditions tested, the small RNA ArcZ is not involved in the upregulation of rpoS expression by DksA. Taken together with the effects of DksAN88I on iraP expression shown in Fig. 2, our results support a role for DksA in both dsrA and iraP expression and thus in posttranscriptional regulation of rpoS.
Effects of DksAN88I on transcription from PiraP and PdsrA in vitro.To address whether the effects of WT DksA and DksAN88I on the dsrA and iraP promoters in vivo were direct, we measured transcription in vitro with purified DksA, ppGpp, and RNA polymerase (Fig. 5). In the presence of ppGpp, WT DksA stimulated transcription from the dsrA promoter as much as 4-fold in vitro (Fig. 5A and C) (37) and from the iraP promoter as much as 6-fold (Fig. 5D and F) (29). Like WT DksA, DksAN88I also stimulated transcription from these promoters in vitro in the presence of ppGpp (Fig. 5B, C, E, and F). However, as with WT DksA on other positively regulated promoters (34), the increased RNAP binding activity of DksAN88I did not relieve the requirement for ppGpp for activation of transcription in vitro (Fig. 5G and H).
Stimulation of PiraP and PdsrA promoter activity by DksAWT or DksAN88I in vitro with or without ppGpp. (A) Representative gel image illustrating fold activation by DksA with or without ppGpp on the dsrA promoter. Transcription was normalized to that in the absence of any factor (lanes 1 and 2), and fold activation for each reaction is shown beneath each lane (average of 2 reactions). Multiple-round in vitro transcription was performed with 10 nM RNAP and 170 mM NaCl at room temperature. Lanes 1 and 2, buffer only; lanes 3 and 4, 2 μM WT DksA only; lanes 5 to 11, 2 μM WT DksA plus ppGpp. The wedge indicates increasing ppGpp, from 3 to 200 μM. Plasmid templates also contained the RNA-1 promoter. Transcripts are indicated by arrows. (B) Representative gel image of transcription from the dsrA promoter as in panel A but with 0.2 μM or 2 μM DksAN88I, as indicated. (C) Maximum activation observed from data sets illustrated in panels A and B plotted relative to transcription in the absence of either DksA or ppGpp. Shown is fold activation with 2 μM DksAWT alone or with DksAWT plus 100 μM ppGpp (black bars), 0.2 μM DksAN88I alone or 0.2 μM DksAN88I plus 25 μM ppGpp (light-gray bars), and 2 μM DksAN88I alone or 2 μM DksAN88I plus 6.25 μM ppGpp (dark-gray bars). The error bars indicate means and SD or range from at least two independent experiments. (D) Same as panel A except the template contained the iraP promoter. (E) Same as panel B except the template contained the iraP promoter. (F) Maximum activation of the iraP promoter from the data sets illustrated in panels D and E plotted relative to transcription in the absence of either DksA or ppGpp. Black bars, 2 μM DksAWT alone or DksAWT plus 100 μM ppGpp; light-gray bars, 0.2 μM DksAN88I alone or 0.2 μM DksAN88I plus 12.5 μM ppGpp; dark-gray bars, 2 μM DksAN88I alone or 2 μM DksAN88I plus 12.5 μM ppGpp. (G and H) Same as panels B and E except the DksAN88I concentration was varied from 4 nM to 4 μM and no ppGpp was included in the reaction mixture (n = 3).
DksAN88I and TraR activate transcription from PiraP and PdsrA better in vivo than in vitro.DksAN88I was identified originally because it allowed growth of a strain lacking ppGpp (ppGpp0; ΔrelA ΔspoT) on media lacking amino acids. DksAN88I inhibited rRNA promoters and stimulated amino acid-biosynthetic promoters in a ppGpp0 strain (34). As with the amino acid-biosynthetic promoters, we found that DksAN88I also stimulated the activities of the iraP and dsrA promoters in a ppGpp0 strain (Fig. 6A and B). Furthermore, the activities of PiraP-lacZ and PdsrA-lacZ fusions after induction of DksAN88I in a ppGpp0 strain background were approximately the same as in the WT background (compare the results in Fig. 6 with those in Fig. 2A and 4B). Thus, even though DksAN88I was unable to activate the dsrA and iraP promoters in vitro without ppGpp (Fig. 5G and H), it was able to activate these promoters strongly in vivo in the absence of ppGpp (Fig. 6A and B) and to turn on the σS stress response (Fig. 2).
DksAN88I and TraR stimulate the dsrA and iraP promoters in the absence of ppGpp in vivo. Overnight cultures were diluted 1:100 in LB medium at 32°C with 0.1 mM IPTG to induce DksAN88I or TraR expression. (A to E) The activities of PiraP-lacZ, PdsrA-lacZ, or PkatE-lacZ fusions were monitored by measuring β-Gal activity during exponential growth at the optical densities shown on the x axes. The cells contained pControl, pDksAN88I, or pTraR. Experiments were performed three times, and representative data are shown. The ppGpp0 strain (ΔrelA ΔspoT) is incapable of synthesizing ppGpp. It was shown previously that TraR can function without ppGpp in vivo (35). (A) Effect of DksAN88I on expression of the PiraP-lacZ fusion. (B) Effect of DksAN88I on expression of the PdsrA-lacZ fusion. (C) Effect of TraR on expression of the PdsrA-lacZ fusion. (D) Effect of TraR on expression of the PiraP-lacZ fusion. (E) Effect of DksA or TraR on expression of the PkatE-lacZ fusion in a WT or ΔrpoS background (open triangles). (F) Western blotting was performed, and σS levels were quantified at different times after TraR induction in a WT or ΔdsrA ΔiraP background as described in Materials and Methods. The error bars indicate SD.
We next examined the effect of TraR on dsrA and iraP expression as an independent means of evaluating the roles of DsrA and IraP in activating the σS regulon. TraR is an RNAP secondary channel binding factor that works without ppGpp but mimics the combined effects of DksA and ppGpp in vivo and in vitro (37, 38). As with DksAN88I, expression of TraR in vivo increased expression of the PdsrA-lacZ and PiraP-lacZ fusions (Fig. 6C and D). Consistent with the model in which increased levels of DsrA and IraP contribute to increased levels of σS, TraR increased σS activity, as monitored by the activity of the σS-dependent PkatE-lacZ transcriptional fusion (Fig. 6E). Correspondingly, TraR increased the levels of σS protein, and these increased levels of σS were not observed in the ΔdsrA ΔiraP strain (Fig. 6F).
However, as observed with DksAN88I by itself, TraR had only a modest effect (∼2- to 2.5-fold) on activating the dsrA and iraP promoters in vitro, smaller than the effects of DksA/ppGpp on the same promoters (Fig. 2; see Fig. S2 in reference 37). The discrepancy between the magnitude of the effects of TraR and DksAN88I on the dsrA and iraP promoters in vitro versus in vivo suggests that part of the effect of DksAN88I and TraR on these promoters in vivo might be indirect. We return to this observation in Discussion below.
DISCUSSION
ppGpp and DksA contribute in multiple ways to increased σS.DksA and ppGpp play crucial roles in the σS response by increasing σS holoenzyme activity (19). We propose that this occurs in at least three different ways. First, DksA and ppGpp together activate the dsrA promoter, thereby improving rpoS translation. Second, DksA and ppGpp together activate the iraP promoter, thereby improving σS stability. Third, we suggest that DksA and ppGpp can increase σS levels by an additional indirect mechanism: by reducing rRNA transcription, DksA and ppGpp increase the availability of RNAP for transcription from other promoters, including σ70-dependent promoters, such as PiraP and PdsrA, and σS-dependent promoters, such as PkatE. We show all three of these contributions of DksA and ppGpp to the σS response schematically in Fig. 7.
Model for stimulation of σS general stress response by DksA/ppGpp. During exponential growth, most RNAP is devoted to transcribing rRNA to accommodate the high demand for ribosomes for protein synthesis. (1 and 2) As nutrients are depleted, either from entry of cells into stationary phase or from starvation for specific nutrients, ppGpp is induced and binds to RNAP, along with DksA, to modulate RNAP activity and activate the dsrA (1) and iraP (2) promoters. (3) At the same time, potent inhibition of rRNA transcription initiation by DksA/ppGpp reduces the level of RNAP transcribing rRNA genes, making more core RNAP available for binding sigma and transcribing from the dsrA and iraP promoters. For brevity, only 4 RNAPs are shown on individual rRNA operons during exponential growth, but well over 100 RNAPs can occupy each of the 7 rRNA operons at high growth rates. Enhanced expression of dsrA and iraP increases rpoS translation and stabilizes σS protein. Thus, DksA/ppGpp upregulates σS expression by at least 3 different mechanisms.
WT DksA can partially inhibit rRNA transcription in the absence of ppGpp (20), but because DksAN88I increases the affinity of DksA for RNAP, it can inhibit rRNA transcription more strongly than WT DksA and bypass the requirement for ppGpp for inhibition. The mechanism of activation by DksA/ppGpp remains poorly understood, but ppGpp binding to the site at the interface of DksA and the β′ rim helices (site 2) is necessary and sufficient for activation (26, 29). How DksAN88I can inhibit rRNA promoters by itself, without ppGpp, but cannot activate promoters without ppGpp remains unclear. We suggest that DksAN88I can cause the conformational change in the promoter-RNAP complex that inhibits transcription but not the one that activates transcription.
The requirement for ppGpp for activation in vitro can be bypassed in vivo.We were able to uncover the third mechanism in the absence of confounding effects of ppGpp because rRNA transcription represents a large fraction of total transcription in vivo during log-phase growth and DksAN88I can inhibit rRNA transcription without ppGpp both in vivo and in vitro. We suggest that the inhibitory effects of DksAN88I and TraR increase the free RNAP concentration in vivo, and this allows transcription from a set of stress-related promoters that are otherwise too weak to be expressed.
We note that activation of weak promoters in this model is not limited to the σ70-dependent promoters PiraP and PdsrA or to σS-dependent promoters like PkatE. Furthermore, effects on unknown factors or solution conditions (pH, salt, and template conformation) could also allow DksA to activate specific promoters in the absence of ppGpp. We note that effects of DksA independent of ppGpp or sRNAs have been reported previously in vivo (39, 40).
We have attempted to compare the contributions of direct activation (as observed with ppGpp/DksA in vitro) with the indirect activation proposed to occur on the same promoter in cells. Although we found they are roughly comparable, these are not ideal comparisons, at least in part because ppGpp concentrations are constantly changing and because the solution conditions in vitro are very different from those in cells. Therefore, we caution against placing too much emphasis on such comparisons.
Effects of DksA/ppGpp on stress responses involving different σ factors.DksA and ppGpp are best recognized for their central roles in the stringent response, largely through their effects on the holoenzyme containing the housekeeping sigma factor σ70. We showed here that DksA and ppGpp also help activate the σS response indirectly by directly stimulating the iraP and dsrA promoters, which in turn results in an increase in rpoS synthesis/stability. DksA and ppGpp have also been shown to regulate the σE regulon directly, a stress response to extracytoplasmic and cell envelope stressors (39, 41). Finally, DksA and ppGpp have been implicated in controlling transcription dependent on σ54 (42, 43). Although some of the effects of DksA and ppGpp are indirect, it appears that they are truly global in scope, affecting regulons controlled by many or most of the cell's sigma factors, allowing the bacterium to survive diverse environmental challenges.
Prospects.Previous studies have linked ppGpp/DksA and sRNA expression to increased σS levels (18, 19), but our work describes the step-by-step chain of events from ppGpp production to increased σS activity. A major component of this signal transduction pathway is the direct activation of a promoter for a small RNA, DsrA. It will be of interest to explore whether ppGpp/DksA regulation of additional sRNAs contributes to the breadth of the ppGpp/DksA regulon, the stringent response, and adaptation to stress.
MATERIALS AND METHODS
Media and bacterial growth.All strains were grown at 32°C with aeration in LB broth supplemented as required with the following: carbenicillin (50 μg ml−1), kanamycin (30 μg ml−1), chloramphenicol (12.5 μg ml−1), and IPTG (isopropyl-β-d-thiogalactopyranoside) (0.1 mM). Plasmids were introduced using standard transformation techniques, and chromosomal alleles were moved into strains via P1 transduction (44). Further details on the methods for strain construction have been described previously (34, 38). All plasmids and strains with their genotypes are listed in Table S1 in the supplemental material.
β-Galactosidase activity assays.Overnight cultures were diluted 1:100 in LB containing carbenicillin, supplemented with 0.1 mM IPTG as needed for induction. Samples (0.5 ml) were taken approximately at every increase of the OD600 by 0.1 until early stationary phase and assayed for β-galactosidase (β-Gal) activity as described previously (44). The graphs are plotted as β-Gal activity per milliliter [OD420 × (2 × 103)/reaction time] versus OD600, and a line of best fit was determined with GraphPad Prism. A representative sample is shown for at least three independent experiments.
Measurement of σS protein via Western blot analysis.Overnight cultures were diluted 1:100 in LB plus carbenicillin, with 0.1 mM IPTG as required for induction, taking 0.9-ml samples at various OD600 values (0.2, 0.4, 0.6, and 0.8), which were then added to 100 μl ice-cold 50% trichloroacetic acid (TCA) to precipitate protein. Samples were pelleted and resuspended at an OD600 of 0.25 in SDS sample buffer (14 μl β-mercaptoethanol, 1 ml 2× SDS buffer, 1 ml elution buffer [10 mM Tris-HCl, pH 8.0]). The same volume of cells from samples at an OD600 of 0.25 was loaded on all lanes and run on an SDS-12% PAGE gel, probed for σS with a polyclonal rabbit antibody (a gift from S. Wickner, NIH), and labeled with Alexa Fluor 647–goat anti-rabbit IgG (Invitrogen) as the secondary antibody. A Typhoon Tri scanner (GE) was used to visualize the bands under the following conditions: emission (Em) filter parameter of 670BP 30 Cy5; voltage sensitivity (PMT) of 300. The bands were then quantified using ImageQuant TL software and graphed for at least three experiments, with bands from one representative Western blot shown. To ensure proper loading, contrast was enhanced in ImageQuant TL to visualize the background banding patterns for consistency across wells.
In vitro transcription.Multiple-round in vitro transcription was performed by standard procedures as previously described (20, 26). DksAWT and DksAN88I were purified as described previously (34). The plasmid templates contained the promoter PdsrA (pRLG13072) or PiraP (pRLG11350) and the transcription terminators rrnB T1 and T2 downstream from the promoter fragment cloning site. DksAWT and DksAN88I concentrations are shown in Fig. 5. The ppGpp concentrations were 3, 6.25, 12.5, 25, 50, 100, and 200 μM (indicated as wedges in Fig. 5). ppGpp was obtained from TriLink Inc. Transcription was quantified by phosphorimaging using ImageQuant 5.2 software, normalized to transcription in the absence of factors, and graphed for each condition. Transcription experiments were performed at least twice.
Statistics.Data were graphed using GraphPad Prism 5 software. The error bars indicate means and standard deviations (SD) for the indicated number of replicate experiments.
ACKNOWLEDGMENTS
We thank Nadim Majdalani and Susan Gottesman (NIH) for kindly providing the parent lacZ fusion strains and Sue Wickner (NIH) for the anti-σS antibody. Additionally, we thank Jamie Catanese and David Marciano for advice on Western blot techniques, as well as Alasdair Gordon, Dominik Satory, and Priya Sivaramakrishnan for providing helpful comments and discussions regarding manuscript preparation.
This work was funded by NIH 1R01GM088653 to C.H., NIH R01 GM37048 to R.L.G., and NSF fellowship NSF DGE-1255980 (NSF 0440525) to M.E.G.
FOOTNOTES
- Received 2 August 2017.
- Accepted 17 October 2017.
- Accepted manuscript posted online 23 October 2017.
- Address correspondence to Christophe Herman, herman{at}bcm.edu.
M.E.G. and S.G. contributed equally to this work.
Citation Girard ME, Gopalkrishnan S, Grace ED, Halliday JA, Gourse RL, Herman C. 2018. DksA and ppGpp regulate the σS stress response by activating promoters for the small RNA DsrA and the anti-adapter protein IraP. J Bacteriol 200:e00463-17. https://doi.org/10.1128/JB.00463-17.
Supplemental material for this article may be found at https://doi.org/10.1128/JB.00463-17.
REFERENCES
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