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. 2021 Oct 27;16(10):e0259067. doi: 10.1371/journal.pone.0259067

The secondary messenger ppGpp interferes with cAMP-CRP regulon by promoting CRP acetylation in Escherichia coli

Chunghwan Ro 1, Michael Cashel 1, Llorenç Fernández-Coll 1,*
Editor: Benjamin J Koestler2
PMCID: PMC8550359  PMID: 34705884

Abstract

The cAMP-CRP regulon coordinates transcription regulation of several energy-related genes, the lac operon among them. Lactose, or IPTG, induces the lac operon expression by binding to the LacI repressor, and releasing it from the promoter sequence. At the same time, the expression of the lac operon requires the presence of the CRP-cAMP complex, which promotes the binding of the RNA polymerase to the promoter region. The modified nucleotide cAMP accumulates in the absence of glucose and binds to the CRP protein, but its ability to bind to DNA can be impaired by lysine-acetylation of CRP. Here we add another layer of control, as acetylation of CRP seems to be modified by ppGpp. In cells grown in glycerol minimal media, ppGpp seems to repress the expression of lacZ, where ΔrelA mutants show higher expression of lacZ than in WT. These differences between the WT and ΔrelA strains seem to depend on the levels of acetylated CRP. During the growth in minimal media supplemented with glycerol, ppGpp promotes the acetylation of CRP by the Nε-lysine acetyltransferases YfiQ. Moreover, the expression of the different genes involved in the production and degradation of Acetyl-phosphate (ackA-pta) and the enzymatic acetylation of proteins (yfiQ) are stimulated by the presence of ppGpp, depending on the growth conditions.

Introduction

The second messengers (p)ppGpp (Guanosine pentaphosphate and Guanosine tetraphosphate) have long been known to accumulate and to alter gene expression as a response to nutritional and physical stress throughout the bacterial kingdom [1, 2]. Increased levels of ppGpp will stop bacterial growth, inhibiting the production of RNA, DNA and proteins [3–5]. Maintaining the balance of ppGpp is crucial; too much inhibits growth and too little makes the cell more vulnerable to nutritional stress [6].

In Escherichia coli, the levels of ppGpp depend on the balance between synthesis activity of RelA and the bifunctional activity of SpoT, which contains a weak synthetase and a hydrolase domain. The synthetase of RelA is activated by binding to ribosomes and sensing amino acid starvation when uncharged tRNA binds to ribosomal A sites [7–9]. In contrast, SpoT-dependent accumulation of ppGpp is promoted by carbon source, fatty acid, iron, nitrogen, phosphate starvation, pH and oxidative stress [10–16]. The balance between synthesis and hydrolysis in SpoT can be tilted towards one or the other through the binding of different proteins to SpoT, such as acyl carrier protein, Rsd or YtfK [17–19].

In Gram-negative bacteria, ppGpp regulates transcription initiation by binding directly to RNA polymerase at two sites [3, 20, 21]. The discriminator sequences, located between the promoter -10 and +1 regions, determines whether ppGpp stimulates or inhibits transcription, depending on whether it is AT-rich or GC-rich respectively [20, 22, 23]. Alternatively, ppGpp can directly bind to proteins to alter their catalytic activities [24–26].

The secondary messenger ppGpp is also known to be important for the proper regulation of genes during diauxic growth [27, 28]. This phenomenon, discovered by Monod in 1947 [29], occurs when bacteria are exposed to limiting amounts of glucose and an excess of a less efficient carbohydrate (such as lactose). Under these circumstances bacteria will utilize exclusively glucose until it runs out. Then, cells will stop growing (diauxic lag time) and they alter their gene expression pattern to allow lactose-fueled growth. The level of ppGpp reaches a peak during the diauxic lag time, before resuming growth utilizing lactose [28]. Although several master regulators control diauxic shift [30, 31], ppGpp affects the length of the diauxic lag time by increasing the levels of acetyl-phosphate and, consequently, the levels of acetylated proteins [28].

In Escherichia coli, the phosphotransferase system (PTS) plays a major role in the control of diauxic growth. The preferential glucose uptake is accompanied by its phosphorylation to glucose-6P by the PTS, using phosphoenolpyruvate (PEP) as a phosphate donor. The PTS components will oscillate between their phosphorylated and non-phosphorylated forms. The presence of glucose in the media will promote a continued formation of glucose-6P that will favor the conversion of the PTS components to their non-phosphorylated forms. The non-phosphorylated form of EIIAglc (one of the components of the PTS) binds and inhibits the transport machinery for non-PTS sugars (such as lactose), while inhibiting the synthesis of cAMP. In the absence of glucose, the PTS components remain phosphorylated and can activate the adenylate cyclase synthesis of cAMP [30, 31]. The cAMP receptor protein (CRP) complexed with cAMP, plays a key role in gene expression in Escherichia coli. The cAMP-CRP complex binds to specific sites within the promoter regions of several operons, stimulating or inhibiting transcription initiation [32, 33]. When bacteria grow at a constant temperature without starvation, all cellular components are synthesized exponentially. When the balanced growth rates are determined not by limiting nutrient abundance but by the ability of the cell to use different nutrients in excess, a correlation of the amount of protein, DNA and RNA with the bacterial growth rate is observed [34]. As previously mentioned, this correlation depends on the presence of ppGpp [4, 5]. Using similar conditions, an inverse correlation was found between the levels of (p)ppGpp and growth rate, where higher levels of (p)ppGpp correlate with lower growth rates [35]. In a similar fashion, the expression of carbon catabolic genes (such as lacZ) is inversely proportional to growth rates upon limitation of carbon sources [36]. The levels of the catabolic growth seem to correlate with the levels of cAMP. By controlling the influx of carbon sources, cAMP controls the synthesis of precursors of amino acids that would otherwise control the bacteria proteome [36]. It is interesting to note that limiting the amount of amino acid precursors will promote the RelA-dependent synthesis of ppGpp [28, 37].

As previously mentioned, increasing the levels of ppGpp will negatively affect the growth rate [3]. A recent study [38] has shown that decreasing bacterial growth rate by artificially increasing the levels of ppGpp (by overexpressing a truncated RelA protein with constitutive ppGpp synthesis) results in increasing levels of catabolic enzymes. The study shows a similar inverse correlation between growth rate and expression of carbon catabolic genes as observed when the growth rate is determined by using different carbohydrates, that is attributed to cAMP [36]. A question arises as to whether there is a crosstalk between both secondary messengers, ppGpp and cAMP. Here we use the catabolic gene lacZ as a reporter of the effects that ppGpp may exert over the cAMP-CRP complex. We explore the possibility that ppGpp controls the ability of CRP to bind to the promoter region. Considering that CRP can be acetylated by acetyl-phosphate, changing its ability to bind DNA [39], and that ppGpp controls the levels of acetyl-phosphate [28], we suggest that ppGpp may alter the acetylation state of CRP.

Materials and methods

Strain construction and growth media

All strains and plasmids used are listed in S1 Table. The different strains were constructed by P1 transduction of gene deletions that came from the Keio collection (marked with kanamycin resistance cassette) or from BW16470 (ΔackA Δpta zej223::Tn10). M9 minimal media was prepared as previously described [4], supplemented with either glucose 0.2%, 0.4% lactose, 0.4% glycerol or 0.2% of N-acetylglucosamine (NAG), when indicated. The following antibiotics were added at the following concentrations: 15 μg/ml tetracycline (Tc) and 20 μg/ml kanamycin (Km). IPTG (Isopropyl-β-D-thio-galactopyranoside) was added to a final concentration of 1 mM.

β-galactosidase assays

Two independent cultures (25 ml in 125 ml flasks) for each strain were grown in minimal media with aeration at 37°C to an OD600 of 0.1, supplemented with IPTG 1 mM. 1 ml samples were harvested, placed in ice and β-galactosidase activity determined as previously described in [40]; using ortho-nitrophenyl-β-galactoside (ONPG) as a substrate, and the Miller Units are calculated as (1000 x (OD420−1.75 x OD550)) / (time x volume x OD600).

Western blots

Cells were grown by duplicate in minimal media with aeration at 37°C up to an OD600 of 0.1. Then,1 ml culture was centrifuged, and 1X Sample Buffer (Thermo Fisher) was added to the cell pellet. Samples were boiled and resolved on a NuPAGE 10% Bis-Tris Gel in MES SDS Running Buffer from Thermo Fisher. Proteins were transferred onto a nitrocellulose membrane with an iBlot gel transfer system. Crp and RpoA were detected with α-Crp, α-RpoA mouse antibody (Biolegend) diluted 1/5000 in PBS-T with 2.5% of Milk. To detect lysine-acetylated proteins mouse anti-AcK antibody (Cell Signaling Technology) diluted 1/1000 in TBS-T with 5% of BSA was used. IRDye 800CW Donkey anti-mouse (Li-Cor) was used as secondary antibody. The signal was detected with Odyssey CLX System from Li-Cor, and the intensity of the different bands was quantified using the software Image Studio Lite from Li-Cor.

cAMP quantification

The levels of cAMP (Cyclic adenosine monophosphate) were determined through the usage of cAMP-Glo™ Max Assay kit (Promega, cat. #V1681). Briefly, cells were grown by duplicate in minimal media (50 ml of media in 250 ml flasks) with aeration at 37°C to an OD600 of 0.1. 50 ml were harvested by centrifugation and resuspended with Induction Buffer provided in the kit. Samples were then processed as indicated by the manufacturer. This commercial kit is based on the principle that cAMP stimulates protein kinase A activity; this stimulation depletes ATP in the reaction resulting in decreased light production in a coupled luciferase reaction. Luminescence was then detected with a Synergy HT plate reader and the amount of cAMP adjusted by the culture’s A600.

ppGpp measurement

The levels of ppGpp were measured by Thin Layer Chromatography (TLC) after labelling with P32 as previously described in [41].

Diauxic shift measurement

Cells were grown in M9 minimal media supplemented with 0.025% of glucose and 0.4% of lactose, in a 96-well plate at 37°C with aeration. OD600 was measured every 10 minutes for a total of 12 hours with a Synergy HT plate reader. The Diauxic lag time was determined as described in [28].

Gene expression by RT-qPCR

Expression of cyaA, pta, ackA, yfiQ and cobB were determined by RT-qPCR. Briefly, 2 independent cultures for each strain were grown up to OD600 0.1, when samples were subject to RNA isolation with Trizol (ThermoFisher) as indicated by the manufacturer. The RNA samples were retrotranscribed into cDNA using the high-capacity cDNA reverse transcription kit from Applied Biosystems and target genes were amplified with SYBR Green PCR Master Mix from Applied Biosystems in a LightCycler 480 instrument. The relative gene expression was determined with the comparative CT method, as described in [42] with 3 technical replicates (6 values for each experimental condition). The scpA gene was used as an internal control, since it is not affected by either ppGpp or CRP [27], compared to other house-keeping genes commonly used (such as rRNA operons, gyrA, parC, zwf or gapA). Primers used in this study are listed in S2 Table.

Results

ppGpp interferes with the CRP-cAMP regulon

Transcription initiation from the lacZ promoter is negatively regulated by LacI and is positively regulated by CRP-cAMP. As previously mentioned, the expression of carbon lacZ is inversely proportional to growth rates, when the growth rates are determined by using different carbohydrates [36]. In this study, You et al. used 1 mM IPTG to ensure that LacI will not bind to the lacZ promoter, giving a similar behavior as using a LacI-deficient background. As a consequence, the expression of lacZ is exclusively dependent on CRP-cAMP, which becomes a reporter for this complex [36]. Considering the role of ppGpp controlling the growth rate [3–5], we wanted to determine if ppGpp has any effect over this correlation.

The strain MG1655 (WT) was grown in M9 minimal media with different carbon sources up to exponential phase, and then samples were taken to determine lacZ expression (by measuring β-galactosidase activity). The ability to use each carbon source determines the growth rate of each sample. Then, the lacZ expression was correlated to the growth rate (Fig 1A). As previously described [36], the expression of lacZ seems to inversely correlate with the growth rate, where slower growth rates show higher β-galactosidase activity.

Fig 1. ppGpp affects lacZ expression dependent on growth rate.

Fig 1

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MG1655 (WT) and CF18005 (ΔrelA) were grown up to exponential phase (OD600 of 0.1) in M9 minimal media supplemented with glucose (squares), glycerol (diamonds), lactose (triangles) and NAG (circles). The lacZ expression was measured from 2 biological replicates and 3 technical replicates. The levels of lacZ expression levels were normalized to the expression of WT strain in M9 with glucose. Error bars represent SD. Lmax was calculated as in [36].

Strains deficient in ppGpp (ΔrelA ΔspoT) do not grow in minimal media without amino acids [10], therefore we used a strain lacking the RelA synthetase. RelA-dependent synthesis of ppGpp responds to starvation of amino acids [43], but also it can sense carbon source starvation in the absence of amino acids [28, 44]. Therefore, under the tested conditions, most of the ppGpp will be synthesized by RelA. We have measured the levels of ppGpp in minimal media supplemented with either glucose or glycerol, and we observe, as expected, that most of the ppGpp significantly decreases in the absence of relA (S1 Fig). The small amounts of ppGpp detected in absence of RelA, is produced by SpoT, which is sufficient to keep the cells alive under this conditions. As previously mentioned, the levels of ppGpp inversely correlates with the growth rate [35]. Thus, we observe higher levels of ppGpp in glycerol than in glucose in the WT strain (S1 Fig).

As observed for WT, the lacZ expression in ΔrelA strain is inversely proportional to the growth rate, but with a different slope than WT (Fig 1A). We observe that cells growing on M9 minimal media supplemented with glucose (Fig 1A, squares), display the fastest growth rate but they show no differences between WT and ΔrelA. However, with cells growing slower in glycerol (Fig 1A, diamonds), the ΔrelA strain shows significantly higher lacZ expression than WT (Fig 1A). The absolute values of β-galactosidase activity from the WT and ΔrelA strains grown in glucose and glycerol can be found in Fig 1B. The maximum beta-galactosidase activity (Lmax, intersection with X-axis as described in [36]) is the highest (theoretical) β-galactosidase activity that these strains can reach. The Lmax was measured for each strain and added to Fig 1A. While the WT strains can reach maximum β-galactosidase activity of 2.58 times the activity in glucose, the ΔrelA mutants can reach a maximum β-galactosidase activity of 4.10 times the activity of WT in glucose.

The first question is whether ppGpp can affect the expression of lacZ by directly affecting the promoter expression or indirectly by affecting the CRP-cAMP complex. As previously mentioned, genes directly regulated by ppGpp show a GC-rich discriminator (region between -10 box and +1) if repressed by ppGpp or an AT-rich discriminator if stimulated [20]. Neither lacUV5 nor lac promoter show such discriminator, suggesting that the any effect is indirect. Moreover, In vitro experiments have previously shown that ppGpp affects the expression of the lac operon but only in the presence of the CRP binding site [45, 46], suggesting that any effect of ppGpp over the lac operon requires CRP-cAMP. As corroboration, we determined the expression of the promoter lacUV5, (using a multicopy plasmid with a lacUV5 promoter, which lacks the CRP binding site, fused to RFP-coding gene) in the presence or absence of relA. As previously described in vitro [46], the lac operon does not respond to the presence or absence of relA in absence of the CRP binding site (S2 Fig). These experiments discard the possibility of ppGpp directly affect the lac promoter. Instead, ppGpp indirectly affects the expression of lacZ by affecting CRP or cAMP.

The effect of ppGpp over lacZ depends on acetylation of CRP

We think that ppGpp may affect the CRP-cAMP regulon in 3 possible ways: 1) affecting the levels of cAMP, 2) affecting the levels of CRP or 3) affecting the activity of CRP. Then, the levels of CRP and cAMP were determined under the same conditions as before, in minimal media supplemented with glucose or glycerol (S3 Fig). Despite reports that (p)ppGpp inhibits promoter 2 of crp under stress conditions [47], we find no difference in the levels of CRP between WT and ΔrelA (S3A Fig). At the same time, no significant differences were observed in the levels of cAMP between WT and ΔrelA (S3B Fig), nor in the expression of the adenylate cyclase (cyaA) responsible for producing cAMP (S4 Fig).

Then, since ppGpp does not affect the levels of CRP and cAMP under our conditions, we decided to determine whether ppGpp was affecting the activity of CRP. As previously mentioned, increased ppGpp is associated with increased acetyl-phosphate [28] and the activity of CRP can be modified by its acetylation with acetyl-phosphate [39]; therefore, it seemed possible that ppGpp can affect the acetylation of CRP.

As before, we measured lacZ expression by measuring β-galactosidase activity, but this time using a double mutant deleting the ackA-pta operon, responsible for synthesis and degradation of acetyl-phosphate. We grew WT and ΔrelA with or without the ackA-pta operon in M9 minimal media supplemented with glycerol, and the β-galactosidase activity was measured (Fig 2A). In the presence of the ackA-pta operon, the mutant ΔrelA showed increased lacZ expression compared to WT, as before (Fig 1). However, in absence of ackA-pta, there is no difference in the lacZ expression between the strains with or without relA, suggesting that the differences in lacZ expression produced by ppGpp depend on the presence of acetyl-phosphate and the levels of acetylated proteins.

Fig 2. ppGpp affects lacZ expression by promoting CRP acetylation.

Fig 2

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(A) β-galactosidase activity measure from samples of MG1655 (WT) and CF18005 (ΔrelA) and their isogenic ΔackA-pta mutants grown up to exponential phase (OD600nm 0.1) in M9 minimal media supplemented with glycerol 0.4%. Error bars show standard deviation of 2 biological samples and 3 technical replicates. (B) As before, WT and ΔrelA strains, together with their isogenic ΔyfiQ and ΔcobB mutants were grown in M9 minimal media supplemented with glycerol 0.4% up to exponential phase (OD600nm 0.1) and β-galactosidase activity was measured. Error bars show standard deviation of 2 biological samples and 3 technical replicates. (C) The amount of acetylated CRP, relative to the WT stain, was measured by Western blot from total extracts of WT and ΔrelA strains grown in M9 minimal media supplemented with glycerol 0.4% and grown up to exponential phase (OD600nm 0.1). Western blots and quantification are presented in S6 Fig. Error bars show standard deviation of 2 biological samples. Statistical significance was measured with T-student test (*p-value < 0.05, n.s. p-value > 0.05).

Apart from the non-enzymatic acetylation of proteins produced by acetyl-phosphate, proteins can be acetylated by Nε-lysine acetyltransferases (KATs). In Escherichia coli, the main KAT is YfiQ (also known as PatZ or Pka), although other KATs exist [48]. Instead, CobB is responsible for deacetylation of proteins, independently of the method of acetylation [49]. Then, we decided to also determine the lacZ expression in presence or absence of yfiQ or cobB (Fig 2B). In absence of YfiQ, the expression differences between WT and ΔrelA strains also disappear (Fig 2B), as seen in ΔackA-pta mutants (Fig 2A), suggesting that both enzymatic and non-enzymatic acetylation may be involved. In absence of CobB, we observe a general decrease on the levels of lacZ expression in both strains (Fig 2B), but with a larger difference between WT and ΔrelA. In absence of CobB, the amount of acetylated CRP is predicted to increase, accounting for the general decrease on lacZ expression, and presumably there would be more acetylated CRP in WT strains than ΔrelA strains.

As previously mentioned, ppGpp affects the length of the diauxic lag time by changing the levels of acetyl-phosphate [28]. There, we observed that ΔrelA mutants had longer diauxic lag times than WT in the presence of the ackA-pta operon, but they showed similar diauxic lag times in its absence. Considering that YfiQ seem to be required for the effect of ppGpp over the lacZ expression (Fig 2B), we wanted to determine if YfiQ is necessary for the effects of ppGpp on the diauxic lag time (S5 Fig). No difference was observed in the diauxic lag time in presence or absence of YfiQ (S5 Fig).

Then, we decided to determine the relative amount of acetylated CRP by Western blot in the presence or the absence of RelA (Fig 2C, S6 Fig). Once the detected intensities were normalized to the levels of CRP, the amount of acetylated CRP in a ΔrelA strain is 2-fold lower than WT (Fig 2C). This seems to suggest that ppGpp stimulates the acetylation of CRP under the conditions tested. As predicted, when the amount of acetylated proteins was measured in absence of CobB, the levels of acetylated CRP increased in both WT and ΔrelA strains (S6 Fig).

YfiQ is required for acetylation of CRP promoted by ppGpp

We also measured the amount of acetylated CRP in the absence of acetyl-phosphate (ΔackA-pta) or in the absence of YfiQ in cells grown in M9 minimal media supplemented with glycerol (Table 1). It can be observed that in the absence of the ackA-pta operon, there is a slight decrease on the amount of acetylated CRP, but in the mutant ΔackA-pta ΔrelA we observe even less acetylated CRP than in a ΔackA-pta mutant, suggesting that acetyl-phosphate is not responsible for the acetylation of CRP promoted by ppGpp. Interestingly, in the absence of ackA-pta, the expression of CRP seems to be dependent of ppGpp. A slight decrease on the levels of CRP is observed in the mutant ΔackA-pta ΔrelA (Table 1). If we consider that acetylated CRP would not bind to the promoter region, then we can normalize the amount of CRP to the amount of acetylated CRP (ratio of CRP/Ac-CRP, Table 1). We observe that both strains (ΔackA-pta and ΔackA-pta ΔrelA) show similar ratios of what we may call “active CRP”, which could account for the similar lacZ expression observed before (Fig 2A).

Table 1. YfiQ is required for CRP acetylation.

Amount of CRP acetylated relative to WT in absence of acetyl-phosphate and the KAT YfiQ was measured in M9 media supplemented with glycerol. Western blot can be found in S7 Fig.

Acetylated CRP1 Amount of CRP2 CRP/Ac-CRP3
WT 1.00 ± 0.07 1.00 ± 0.04 100%
ΔackA-pta 0.75 ± 0.08 1.15 ± 0.02 153%
ΔackA-pta ΔrelA 0.45 ± 0.01 0.70 ± 0.04 155%
ΔyfiQ 0.46 ± 0.04 0.90 ± 0.01 194%
ΔyfiQ ΔrelA 0.52 ± 0.02 0.98 ± 0.17 188%
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1 The amount of acetylated protein was normalized to the amount of CRP protein detected in each sample.

2The CRP protein detected by Western blot is normalized to the amount of RpoA detected in each sample.

3 This ratio normalizes the amount of CRP to the number of acetylated CRP.

When we measure the relative amount of acetylated CRP (again, normalized to CRP) in a ΔyfiQ background are 2-fold lower than WT (Table 1), but independent of the presence or the absence of RelA, where the number of acetylated CRP does not change comparing ΔyfiQ with ΔyfiQ ΔrelA. We interpret these results as the effect of ppGpp over CRP acetylation seems to require the presence of YfiQ, during growth in minimal media supplemented with glycerol.

The expression of pta, ackA and yfiQ depends on ppGpp

Finally, we wanted to determine the effect ppGpp on the gene expression of the ackA-pta operon, yfiQ and cobB by qPCR (Fig 3).

Fig 3. Effect of ppGpp on gene expression of the main factors contributing to protein acetylation.

Fig 3

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The gene expression of (A) pta, (B) ackA, (C) yfiQ and (D) cobB were measured by qPCR from WT and ΔrelA cells grown in minimal media with glucose or glycerol. Expression levels are normalized to the expression in WT grown in glucose. Error bars show standard deviations of 2 biological samples and 3 technical replicates. Statistical significance was measured with T-student test (*p-value < 0.05, n.s. p-value > 0.05).

The expression of pta show an almost 2-fold decrease in the absence of relA compared to WT in minimal media with glucose (Fig 3A), suggesting that the expression of pta depends on the presence of ppGpp. However, in minimal media with glycerol, we observe that the expression of pta is repressed in both strains, and the difference between WT and ΔrelA is rendered non-significant. Instead, the expression of ackA (Fig 3B) seems to not be affected by ppGpp while growing in glucose. In glycerol we observe a 2-fold decrease in the expression of ackA in absence of relA compared to WT; however, as seen for pta, the expression of ackA is strongly repressed while growing in glycerol compared to glucose.

While the enzymes responsible for producing acetyl-phosphate are repressed in minimal media supplemented with glycerol (Fig 3A and 3B), the expression of yfiQ (Fig 3C) shows a 4-fold stimulation in cells growing in glycerol compared to glucose, consistent with CRP stimulating the expression of yfiQ [50]. Moreover, in absence of relA there is a decrease on the expression of yfiQ in minimal media supplemented with either glucose or glycerol (Fig 3C). Our data suggests that ppGpp stimulates yfiQ expression, probably directly (considering that yfiQ seems to have an AT-rich discriminator). Consistently, it was previously described that the expression of yfiQ increases during stationary phase compared to exponential phase, but does not seem to require σS [50]. Not many differences were observed on the expression of cobB (Fig 3D).

Discussion

As previously shown [36], the gene expression of lacZ inversely correlates with growth rate (Fig 1A), showing lower expression in higher growth rates and higher expression in lower growth rates. This correlation has been described to be cAMP dependent [36]. Here we show that variations on the levels of ppGpp (by deleting relA) affect the slope of such correlation. Considering that the basal levels of ppGpp increases as growth rates decrease [35], it is perhaps not farfetched to suggest a combined effect of cAMP and ppGpp on the expression of lacZ and other catabolic enzymes. In fact, a similar inverse correlation of metabolic enzymes expression with growth rate was observed when the growth rate was decreased by artificially increasing the levels of ppGpp [38].

The effect of ppGpp on lacZ expression is not a direct effect on its promoter, but an indirect effect by controlling the acetylation of CRP. Our data suggest that during growth in minimal media supplemented with glycerol, the acetylation of CRP requires the presence of the acetyltransferases YfiQ (Fig 2B, Table 1). Both, acetyl-phosphate and YfiQ, have been shown to have the potential to acetylate certain CRP residues [51]. In that study, Kuhn et al. explore the different acetylated proteins through mass spectrometry using a WT strains and different mutants (ΔyfiQ among them). As they show at their supplementary material, the residue 27 of CRP seems to be acetylated in the WT strain but not in the mutant ΔyfiQ. Evidently, further studies will be required to determine what residues of CRP are acetylated by YfiQ in a ppGpp-dependent manner, and how it does impair lacZ transcription.

As previously described, the levels of acetyl-phosphate seem to be dependent on ppGpp during growth in glucose [28], consistent with ppGpp stimulating the expression of ackA-pta operon in minimal media with glucose (Fig 3). Under the same conditions, acetyl-phosphate can acetylate residue K100 of CRP, modifying its ability to bind to Class II promoters, but not class I promoters [39]. There are three classes of CRP-dependent promoters, depending on the contact between CRP and the α subunit of RNAP [52]. Consequently, ppGpp has no effect on the expression lacZ (a class I promoter) in minimal media with glucose. In minimal media with glycerol, the expression of the operon ackA-pta is low (Fig 3), suggesting that the amount of acetyl-phosphate is probably low too. Instead, the levels of YfiQ dramatically increase in minimal media with glycerol, stimulated by ppGpp (Fig 3), and it can acetylate some residues of CRP, probably different from acetyl-phosphate [51]. The acetylation of CRP by YfiQ seems to be responsible for the effect of ppGpp on lacZ expression (Fig 2B, Table 1), suggesting that the residues acetylated by YfiQ may affect class I promoters (at least the lac operon). Then, we could hypothesize that while the acetylation of CRP by acetyl-phosphate during growth in glucose will affect the expression of class II promoters, the acetylation of another residue by YfiQ during growth in glycerol may affect the expression of class I promoters. Thus, by changing the acetylating state of CRP, or even the residues acetylated, ppGpp may modify the expression levels of a subset of genes regulated by CRP depending on the carbohydrate available.

Despite that deleting the ackA-pta operon shows a similar effect over the role of ppGpp on the lacZ expression than deleting yfiQ (Fig 2A and 2B), acetyl-phosphate does not seem to be responsible for the acetylation of CRP promoted by ppGpp in cells grown in minimal media supplemented with glycerol (Table 1). This is also consistent with the expression pattern observed (Fig 3), where the ackA-pta operon is repressed in media supplemented with glycerol.

As previously discussed, in vitro experiments using cell-free extracts–grown in media with amino acids–seem to suggest that ppGpp has an effect over the expression of the lac operon but only in the presence of the CRP binding site [45, 46]. Instead, similar experiments performed during amino acid starvation (by addition of pseudomonic acid) show a RelA-dependent accumulation of ppGpp that strongly inhibits the expression of lacZ [53]. In our case, cells are grown in minimal media without amino acids, and as observed by Little and Bremer [53], we observe that ppGpp exerts a negative effect on lacZ expression (increased expression in absence or RelA). However, our conditions are not as extreme as the amino acid starvation that they produce; hence, we observe a minor inhibition of lac operon. In any case, the effect of ppGpp on lacZ requires the presence of CRP [45] or the CRP binding site (S2 Fig; [46]), suggesting that it is an indirect effect by affecting CRP.

We can also find some discrepancies in the effect of ppGpp on crp expression: while in vitro experiments suggested that ppGpp is able to repress the promoter P2 of crp [47], it seems that ppGpp can positively affect the expression of CRP during severe amino acid starvation [54]. Instead, no effect on the expression of CRP has been observed under the conditions tested here (S3A Fig).

Altogether, our data suggest that ppGpp can modulate the expression of genes regulated by CRP-cAMP by affecting the acetylation state of CRP, and probably, modifying its ability to bind. It is still to be determined how many genes from the ppGpp regulon depend on its effect on global protein acetylation, and more particularly, how many are shared with CRP-cAMP by changing the acetylation state of CRP. Transcriptomic studies performed during diauxic shift, show an overlap between the genes regulated by CRP and ppGpp [27], and we believe that some of these overlapping effects could be the result of CRP acetylation promoted by ppGpp. Evidently, new transcriptomic studies that include ΔackA-pta and ΔyfiQ mutant strains need to be performed in future studies.

Supporting information

S1 Fig. Levels of ppGpp.

The amount of ppGpp was measured relative to total amount of G (pppGpp + ppGpp + GTP) by TLC in MG1655 and ΔrelA strains grown in MOPS media with 0.2% glucose or 0.4% glycerol. Error bars represent SD of two biological replicates. Statistical significance was measured with T-student test (*p-value < 0.05, n.s. p-value > 0.05).

(TIFF)

Click here for additional data file. (461.6KB, tiff)
S2 Fig. The effect of ppGpp on lacZ expression requires of CRP binding site.

MG1655 (WT) and CF18005 (ΔrelA) harboring the plasmid pBbA5k were grown in M9 minimal media supplemented with glycerol 0.4% and IPTG 1 mM. Finally, fluorescence emitted by RFP (Red Fluorescent Protein) was measured. The amount of fluorescence was plotted against the OD600, giving a linear correlation where the slope is the RFP production rate per OD600. Error bars represent SD. Statistical significance was measured with T-student test (n.s. p-value > 0.05).

(TIFF)

Click here for additional data file. (378.7KB, tiff)
S3 Fig. ppGpp does not affect levels of CRP or cAMP.

(A) MG1655 (WT) and CF18005 (ΔrelA) were grown in M9 minimal media supplemented with either glucose 0.2% or glycerol 0.4% up to exponential phase (OD600 0.1) and protein levels of CRP and RpoA were measured by Western blot. Means and standard deviation are shown of CRP amounts normalized to the amounts of RpoA (loading control) relative to the values from WT in glucose. (B) Measurements of cAMP of cells grown as in panel (A). Error bars show standard deviation of 2 biological samples. Statistical significance was measured with T-student test (n.s. p-value > 0.05).

(TIF)

Click here for additional data file. (3.9MB, tif)
S4 Fig. The gene expression of cyaA measured by qPCR from WT and ΔrelA in minimal media with glucose or glycerol.

Expression levels are normalized to the expression in WT grown in glucose. Error bars show standard deviation of 2 biological samples and 3 technical replicates. Statistical significance was measured with T-student test and no difference was observed between WT and ΔrelA (p-value > 0.05).

(TIF)

Click here for additional data file. (363.6KB, tif)
S5 Fig. The KAT YfiQ does not affect the diauxic shift.

(a) The strain MG1655 (wt) and ΔrelA, together with their isogenic ΔyfiQ and ΔcobB mutants were grown in M9 with 0.025% glucose and 0.4% lactose for 12 h and OD600 measured every 10 min. Ratios of diauxic time normalized to generation times of three independent experiments with duplicate wells (six values) were plotted as box plots. Bottom and top of the colored box represent first and third quartiles, and the band inside the box is the median. Whiskers represent minimum and maximum data, while circles are outliers (single points). (b) Typical diauxic growth curve from WT strain.

(TIFF)

Click here for additional data file. (1.6MB, tiff)
S6 Fig. ppGpp controls acylation of CRP, Westen blots.

The amount of acylated CRP was measured by Western blot from total extracts of WT and ΔrelA strains together with their isogenic ΔcobB mutants, were grown in M9 minimal media supplemented with glycerol 0.4% up to OD600 of 0.1. To ensure a proper identification of the CRP protein, a Δcrp mutant was also added. The amount of acetylated protein (detected by Western blot using antibody specific for acetylated lysines, Ac-K) was normalized to the amount of CRP and presented relative to the WT strain. STDV = standard deviation.

(TIF)

Click here for additional data file. (4.3MB, tif)
S7 Fig. YfiQ acetylates CRP, Western blots.

The amount of acylated CRP was measured by Western blot from total extracts of the WT strain, together with the ΔackA-pta, ΔackA-pta ΔrelA, ΔyfiQ and ΔyfiQ ΔrelA strains. Cells were grown in M9 minimal media supplemented with glycerol 0.4% up to exponential phase (OD600 0.1). To ensure a proper identification of the CRP protein, a Δcrp mutant was also added. The amount of acetylated protein (detected by Western blot using antibody specific for acetylated lysines, Ac-K) was normalized to the amount of CRP and presented relative to the WT strain. The CRP amounts are normalized to the amounts of RpoA (loading control).

(TIF)

Click here for additional data file. (3.8MB, tif)
S1 Table. Bacterial strains and plasmids used in this study.

(PDF)

Click here for additional data file. (257KB, pdf)
S2 Table. List of primers used in this report.

(PDF)

Click here for additional data file. (133.5KB, pdf)
S1 File. Uncropped and unadjusted Western blot images.

(PDF)

Click here for additional data file. (563.5KB, pdf)

Acknowledgments

We would like to thank Dr. Sankar Adhya (NCI) for providing material necessary for the construction of different strains.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This research was funded by the intramural research program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), NIH. CR recieved a stipend from the NIH Office of Intramural Training & Education. Funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Decision Letter 0

Benjamin J Koestler

13 Jul 2021

PONE-D-21-19172

The secondary messenger ppGpp interferes with cAMP-CRP regulon by promoting CRP acetylation in Escherichia coli.

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Reviewer #1: Nucleotide second messengers (NSMs) are utilized across all domains of life. Bacteria respond to environmental stimuli by increasing second messengers, each of which transmits and amplifies the signal by activating a subset of downstream pathways to optimize appropriate physiological adaptations. Each NSM has multiple but distinct downstream targets. In this nicely written manuscript, Ro et al. address an indirect crosstalk between two second messenger systems. They show that ppGpp interferes with expression of the cAMP-CRP regulon by stimulating expression of the acetyltransferase YfiQ which acetylates CRP in Escherichia coli. This interesting study may contribute to the base of our knowledge on bacterial physiology. I have only one suggestion to offer concerning the physiological role of this regulation.

The data in this study suggest that ppGpp stimulates expression of YfiQ and thereby increases CRP acetylation (Figures 2, 3, & S5). The acetylation of CRP impairs its ability to bind to DNA. Therefore, according to this regulation, ppGpp interferes with lacZ expression. However, as previously known that both the expression of the CRP-cAMP regulon and the levels of ppGpp inversely correlates with the growth rate, authors observed higher levels of beta-galactosidase activities and ppGpp in glycerol than in glucose (Figures 1 and S1). Thus, the higher level of ppGpp appears to correlate with the higher expression of the CRP regulon gene, lacZ, under certain circumstances. Therefore, the physiological significance of the CRP-interfering role of ppGpp needs to be more clearly stated in the discussion session. In what circumstances would this regulation operate?

Minor corrections:

1. L95. N-acetylglucosamine (A in lower case).

2. L97, 1 mM (space).

3. L134. Is there any specific reason why he scpA gene was used as an internal control, instead of a house-keeping gene such a rRNA-coding gene?

4. L137. INTEReFERES.

5. L164. The maximum beta-galactosidase activity?

6. L174. In Fig S2B, RFP, but not lacZ, was used as a reporter gene.

7. L177. acETylation?

8. L261. showS.

9. L289. We could not find that YfiQ has the potential to acetylate certain CRP residues in the reference 50.

Reviewer #2: Manuscript (PONE-D-21-19172): The secondary messenger ppGpp interferes with cAMP-CRP regulon by promoting CRP acetylation in Escherichia coli.

Authors: Ch. Ro, M. Cashel and Ll. Fernández-Coll

In this manuscript Ro et al., investigated the relationship between the alarmone ppGpp, the Catabolite Represor Protein CRP and the second messenger cAMP in regulating the expresión of the lacZ gene from Escherichia coli. Employing genetic, physiologic and immunologic approaches, the authors described that i) ppGpp indirectly affects the expression of lacZ by affecting the CRP-cAMP complex, ii) ppGpp regulates the expression lacZ through CRP-acetylation, iii) YfiQ is responsible of acetylating CRP in a RelA-dependent manner, and finally, iv) RelA regulates in a positive manner the expression of yfiQ. Based on these results, they concluded that ppGpp induce the acetylation of CRP, thus affecting its regulatory properties with a direct impact on the expression of the lacZ gene.

The work addresses a very interesting topic regarding a novel posttranslational mechanism that presumably regulates the transcriptional properties of cAMP-CRP over the Lac operon in E. coli. However, the experimental evidence shown in only 3 Figures in the main text is not enough to support this notion. Below, please find comments and suggestions that the authors may find useful to improve the scientific impact of their manuscript.

Major comments:

1) Lines 138-139: In the experimental approach the authors determined beta-galactosidase levels to evaluate the transcriptional control exerted by cAMP-CRP over the lacZ operon. In these paragraphs they indicate that addition of IPTG (1 mM) eliminate the repressor effects of LacI; however, in most of the experiments the authors don’t indicated if the minimal media employed with different carbon sources was supplemented with IPTG. Furthermore, with such amenable model of study, it calls the attention that the authors did not employ a lacI-deficient strain to unambiguously rule out the repressor effects of LacI.

2) Lines 146-51: The authors justify the advantage of using a RelA-deficient strain instead of a double relA spoT mutant. The rational is correct; however, as shown in Figure S1, in culture media supplemented with glycerol and glucose the relA mutant still contain ppGpp. Can the authors clarify if this amount of the alarmone is influencing or not their results. Perhaps, the use of an available relA spoT strain would render more confident experiments in a genetic background fully depleted of ppGpp. Also, Figure S1 do not show if there are statistical differences in the levels of ppGpp between the strains and carbon sources employed, please include this information in the revised manuscript.

3) As the authors stated in the introduction, the cAMP-CRP complex controls the expression of several catabolic genes; therefore, to better reinforce the conclusions described in this work, the authors must include experiments with reporters additional to lacZ.

4) Lines 165-168. The manner of presenting the results in Figure 1 are rather confuse. This reviewer and the interested audience would be benefited if the authors present in a more detailed form these results. Please move the results of Figure S2-A to the main text with the following considerations:

A) It is not clear the growth phase in which the strains were analyzed; therefore, these plots must show the growth levels by OD600nm and ideally by viable counts.

B) In the same Figure, plot in a time dependent fashion, the levels of beta-galactosidase of the strains analyzed in the distinct carbon sources employed

C) Here, a question arises: what is the functional relationship between RelA and Crp in regulating LacZ levels in a LacI-deficient background? This question can be easily tested in relA crp genetic background but plotting the results in a time dependent manner.

5. Lines 169-176:

5.1. Lines 169-170. Please be more specific in explaining how the authors expect that ppGpp could affect the lacZ promoter; more specifically, is not expected that ppGpp regulates lacZ (RFP in their experiments) expression by binding RNA polymerase? Please see https://doi.org/10.1073/pnas.1819682116

5.2. In the results of Figure S2-B, please make sure to explain:

A) that you are using a PlacUV5-Rfp fusion in an extrachromosomal multi-copy plasmid

B) the experiment does not directly measure lacZ, but RFP expression.

C) with a single experimental time-point is difficult to verify that ppGpp (RelA deficiency) does not affect lac-RFP expression, these experiments must be performed in time-dependent fashion plotting also the growth curve, ideally as indicated in the next comment

D) to avoid effects of gene dosage and more appropriately address these experiments, the authors must have considered to work with a strain carrying a single chromosomal copy of the PlacUV5-lacZ fusion.

6. Lines 178-184. Here, the authors stated that the absence of RelA has no effect on the levels of cAMP-CRP. To support this notion, they, independently, analyzed protein levels of CRP, cAMP concentrations and mRNA levels of cyaA. However, to better support this conclusion the authors are strongly encouraged to determine the levels of the cAMP-CRP complex.

7. CRP acetylation experiments (Fig. 2; Figs. S5, S6 and Table 1)

A) Figure 2. Notation of strains tested in Fig. 2 is confuse, please clearly indicate the genotype of each strain and present the results in the following order: WT, ackA-pta, relA, relA/ackA-pta establishing significant differences among the four strains. Please, describe and interprete these comparisons in the main text of the paper.

B). Figs S5 and S6 and Table 1. With the immunological (Western blotting) approach employed to determine levels of acetylated CRP in the distinct genetic backgrounds, is a little difficult to agree with the conclusion of the authors that relA, ackA-pta, yfiQ and cobB regulate the levels of acetylated CRP. Even in the absence of RelA and YfiQ or RelA and AckA-Pta, the anti Ac-K antobodie detects bands of similar intensity than those observed in the WT strain. Furthermore, the protein band control (which I assume is RpoA) shows different intensities. Importantly, a significant fraction of acetylated CRP is detected in all the genetic strains analyzed making more difficult to interprete these results. Therefore, the authors are strongly encouraged to find alternative strategies to quantitate levels of acetylated CRP for instance by LC–MS/MS analysis.

8. The conclusion that YfiQ is responsible of acetylating CRP must be clearly documented as this is a key evidence of the regulation mechanism of CRP proposed by the authors. Please specifiy if CRP has been described as a target for YfIQ. If not, the authors are encouraged to implement a more specific approach to demonstrate this fact, for example, using in in vitro assays the unacetylated form of pure CRP as a substrate for the purified form of YfiQ.

9. Finally, regarding the conclusion that yfiQ expression is regulated by the levels of ppGpp (Fig. 3), the authors must indicate in the discussion section how the alarmone may exert its regulatory functions.

Minor comments.

1) Lines 98-101: Please describe in more detail this protocol, indicate the substrate used and specify how the Miller Units were determined.

2) Lines 123-126. Please show as a supplementary Figure, a typical growth curve to see the diauxic effect and clearly indicate in this curve the OD600nm at which most of the sample were taken for biochemical and immunological determinations.

3) Figs. S3 and S6. Please describe in the Figure Legends the purpose of the Western Blotting of RpoA in these experiments.

**********

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Reviewer #1: Yes: Yeong-Jae Seok

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PLoS One. 2021 Oct 27;16(10):e0259067. doi: 10.1371/journal.pone.0259067.r002

Author response to Decision Letter 0

20 Sep 2021

We have upload a response to the reviewers containing the comments of the reviewers (in black) and our response (in blue). Here we submit only our response to the reviewers' commets. We also want to thank the reviewers for their comments.

Reviewer #1:

We would like to say that this is a really interesting question and this discussion has been added to the modified version of the manuscript (lines 319-335). As previously said in the manuscript, the levels of acetyl-phosphate seem to be dependent of ppGpp during growth in glucose (Fernandez-coll and Cashel 2018), consistent with ppGpp stimulating the expression of ackA-pta operon in minimal media with glucose (figure 3). Under the same conditions, acetyl-phosphate can acetylate CRP, modifying its ability to bind to Class 2 promoters, but not class 1 promoters (Davis et al. 2018). Consequently, ppGpp has no effect on the expression lacZ (a class 1 promoter) in minimal media with glucose. In minimal media with glycerol, the expression of the operon ackA-pta is low (figure 3), suggesting that the amount of acetyl-phosphate is probably low too. Instead, the levels of YfiQ dramatically increase in minimal media with glycerol, stimulated by ppGpp (figure 3), and it can acetylate some residues of CRP, different from acetyl-phosphate (Kuhn et al. 2014). The acetylation of CRP by YfiQ seems to be responsible for the effect of ppGpp on lacZ expression (figure 2B, Table 1), suggesting that the residues acetylated by YfiQ may affect class 1 promoters (at least the lac operon). Then, we could hypothesize that while the acetylation of CRP by acetyl-phosphate during growth in glucose will affect the expression of class 2 promoters, the acetylation of another set of residues by YfiQ during growth in glycerol may affect the expression of class 1 promoters. It is possible that by changing the acetylating state of CRP, or even the residues acetylated, ppGpp may modify the expression levels of a subset of genes regulated by CRP depending on the carbohydrate available.

Minor corrections:

1. and 2. Changed as suggested.

3. Considering that we were planning to study the effects of ppGpp and CRP over gene regulation, we needed a gene that was not affected by either. According to Traxler et al. 2006, scpA is not affected by ppGpp or CRP. Instead, the rRNA operons and other commonly used house-keeping genes (gyrA, parC, zwf, gapA), are regulated by ppGpp or CRP. This is now explained in the modified version of the manuscript (lines 139-140).

4 and 5 changed as suggested.

6. This is now indicated in the text (line 191).

7. and 8. Changed as suggested

9. At Kuhn et al, the authors determine the amount of acetylated proteins through mass spectrometry of WT strains and different mutants (yfiQ among them). As they show at table S1, residue 26 seems to be acetylated in the WT strain, but not in the mutant yfiQ. This information is now described in the discussion (lines 314-318).

Reviewer #2:

Major comments:

1) As indicated by You et al. 2013, adding 1 mM of IPTG mimics a LacI-deficient background, having no effect on the correlation between lacZ levels and growth rate. Thus we decided to not include it on our manuscript, because we believe that it has been already proven. This is now stressed in the text (lines 147-148).

2) As mentioned in line 157, a mutant ΔrelA ΔspoT will not grow in the conditions used for this study, minimal media without amino acids (Xiao, et al. 1991). That forces us, as well as other researchers in our field, to use the closest strain possible to a ppGpp0 strain: in this case a ΔrelA strain. RelA-dependent synthesis of ppGpp responds to starvation of amino acids, but also it will be stimulated by starvation of precursors of amino acids (responding to different carbon sources). Therefore, under the tested conditions, most of the ppGpp is synthesized by RelA, and consequently, a strain ΔrelA shows almost undetectable levels of ppGpp (fig. S1). This is further discussed in the revised manuscript (lines 157-164). Moreover, the statistical differences have been included at the figure S1, as requested by the reviewer.

3) We agree with the reviewer that more reporter genes would strengthen our point, the more the merrier. However, we also need to stress that lacZ has been a staple to report effects over the CRP complex for decades. Transcriptomic studies have shown an overlap between the genes regulated by CRP and ppGpp (Traxler et al. 2006), and we believe that our results suggest that some of these overlapping effects could be the result of CRP acetylation promoted by ppGpp. Therefore, to address this hypothesis, new transcriptomic studies that include ΔackA-pta and ΔyfiQ mutant strains are required. Evidently, this will be part of a follow up manuscript. This is now discussed in the manuscript (line 359-362).

4):

A) and B) The figure 1 is based on the experiments described by You et al. 2013, as indicated on the text, thus we decided to plot it in the same way. There the authors show a correlation between growth rate (μ) and lacZ expression. Then, we believe that plotting OD600nm and β-galactosidase activity in a time-dependent manner may confuse the reader from the main point of the figure, that the β-galactosidase activity is directly proportional to the growth rate and this can be modified by ppGpp. We agree that Figure S2A will help on the comprehension of the figure, and we have moved it to the main text, as suggested by the reviewer. All the samples for measuring β-galactosidase activity were taken at OD600nm of 0.1. Now, this is also added to the figure legend. The growth levels for each strain are indicated with the growth rates (μ) calculated with the OD600nm.

C) This is an interesting point raised by the reviewer. As previously discussed, You et al. 2013 show that the addition of IPTG mimics a LacI-background, and does not affect the regulation of LacZ levels. So, we consider that addition of 1 mM of IPTG has a similar effect as a LacI-deficient background. As mentioned above, this is now stressed in the manuscript. We also agree with the reviewer that a classic way to determine the involvement of CRP on the regulation of ppGpp on lacZ expression, it would be the use of a Δcrp mutant strain. However, a Δcrp strain does not express lacZ (undetectable β-galactosidase activity) and it does not grow in minimal media supplemented with glycerol. Therefore, we decided to include the experiments using the lacUV5 promoter (that do not contain the CRP-binding site), despite it is just a confirmation of a previous result (Yang et al. 1974).

5. Lines 169-176:

5.1. It has previously shown (Yang et al. 1974) that lacUV5 does not respond to the levels of ppGpp. Actually, it has been used by several authors as negative control in experiments of in vitro transcription when studying ppGpp regulation. Then, the experiments in figure S2 need to be taken as a verification of a previous result. We have stressed this notion in the reviewed version of the manuscript (line 191). As pointed by the reviewer, genes directly regulated by ppGpp show a GC-rich discriminator (region between -10 box and +1) if repressed by ppGpp or an AT-rich discriminator if stimulated. Neither lacUV5 nor lac promoter show such discriminator. This information is now added to the manuscript (lines 185-188).

5.2.

A) and B) This has been mentioned in the modified version of the manuscript (line 191), and the figure legend.

C) We agree with the reviewer that this kind of experiments require more than one time-point, and we apologize for the lack of clarity in the figure. In fact, in figure S2 we are not representing the fluorescence of a single time-point, but the rate of production per OD600nm (slope obtained after plotting fluorescence accumulation vs. OD600nm). This is now mentioned in the modified version. However, considering that this experiment is the verification of something previously described (Yang et al. 1974), we do not think that it is necessary to add extra plots or the growth curve.

D) As previously mentioned, this was a confirmation of a published result (Yang et al. 1974). Thus, we believe that it was well established in the original paper.

6. We agree with the reviewer that we determine the effect of ppGpp over the levels of CRP and cAMP but not the levels of the cAMP-CRP complex. If we mentioned on the text that “ppGpp do not affect the cAMP-CRP complex” it has been for a matter of simplicity. This has been modified in the text accordingly. About the recommendation to measure the levels of the complex, first, we do not have the capacity or the experience to perform such experiment. And second, there is no evidence to suggest that ppGpp can physically interfere with the binding of cAMP to CRP. Despite that ppGpp can bind to some proteins, most of the effects are through changing gene expression. Few protein interactions with ppGpp have been described lately, but none with CRP. There are no evidences either that ppGpp can directly interact with cAMP. It is arguable that the acetylation of CRP could interfere with the formation of the complex with cAMP, but as discussed in the text (lines 317-318), that will have to be determined in future studies.

7. A) The aim of the figure 2 is to show that the effect of ppGpp over lacZ requires the acetylation of CRP. First, we have to clarify that panel A and B show β-galactosidase activity experiments, while panel C show CRP’s acetylation levels only in the presence or the absence of RelA. This is now stressed in the figure legend.

Second, the nomenclature used in this figure is the same as used in the rest of the manuscript to ensure the cohesion of the manuscript. In panel A and B, the presence of RelA is indicated by color (as indicated at the figure legend). Any other genetic background is indicated in the X-axis. In panel C, it is true that the color code is broken (both columns are black), but there is only one category.

Third, the acetylation of CRP in ΔackA-pta and ΔyfiQ mutant strains is shown only in Table1 (discussed below).

B) Figures S6 and S7 contain the Western blots to determine the levels of acetylated CRP shown in Figure 2C and Table 1, respectively. We agree that the anti Ac-K antibody detects bands that, at naked eye, seem to be of similar intensity but, those intensities were normalized to the amount of CRP detected at the same samples. Moreover, the intensities are not measured de visu but through the software Image Studio from Li-Cor. This information is now added to the manuscript (line 113-114). Once the detected intensities were normalized to the levels of CRP, the amount of acetylated CRP in a ΔrelA strain happen to be 2-fold lower than WT, and this is reported in fig. 2C. As we interpret this result, ppGpp seems to stimulate the acetylation of CRP under the conditions tested. In figure S7, showing the Western blots used for the table 1, we are determining the effects of YfiQ and AckA-Pta over the levels of CRP (normalized to RpoA) and the levels of acetylated CRP (normalized to the levels of CRP). Here, we observe that the measured intensities of acetylated CRP (again, normalized to CRP) in a ΔyfiQ background are 2-fold lower than WT, but independent of the presence or the absence of RelA. We interpret these results as the effect of ppGpp over CRP acetylation seems to require the presence of YfiQ, and this is how it is now stated on the text.

Despite that LC-MS/MS will give us quantitative levels of acetylated CRP, the antibody used in this manuscript have been used to obtain relative amounts of acetylated proteins in manuscript previously published by our research group, and other research groups in our field. We have stressed that our results are relative amounts instead of quantitative levels (line 247).

8. Acetylation of CRP, yfiQ-dependent, have been previously documented by Kuhn et al. 2014. This has been further discussed, as requested by reviewer 1 (as can be seen above, lines 314-317). It is true that further studies are required to determine more in detail what residues are acetylatilated in a ppGpp-dependent manner, and how does it impair lacZ transcription. But, as it is now discussed in the modified text (lines 317-318), this will be the focus of future manuscripts.

9. We absolutely agree with the reviewer. As previously said, genes directly stimulated by ppGpp has an A/T rich discriminator. As shown by Castaño-Cerezo et al. 2011, the expression of yfiQ is stimulated in stationary phase, but it does not respond to the alternative sigma factor RpoS, suggesting that any effect must be direct. This is now discussed at the modified version of the manuscript (lines 297-299).

Minor comments.

1) This protocol is a well established standard in our field, but, as suggested by the reviewer, more information has been added.

2) We agree that adding a diauxic growth curve will help to understand some parts of the manuscript; it is now a panel of figure S5. However, we believe that indicating there the OD at which most of the samples were taken will be misleading for the reader, considering that only the experiment shown in figure S5 was made during diauxic shift. Instead, we have stressed in all the figure legends that the samples were taken during exponential phase (at an OD600nm of 0.1).

3) This is now added to the figure legends as suggested by the reviewer.

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Decision Letter 1

Benjamin J Koestler

12 Oct 2021

The secondary messenger ppGpp interferes with cAMP-CRP regulon by promoting CRP acetylation in Escherichia coli.

PONE-D-21-19172R1

Dear Dr. Fernández-Coll,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Manuscript PONE-D-21-19172R1.

The secondary messenger ppGpp interferes with cAMP-CRP regulon by promoting CRP acetylation in Escherichia coli.

Authors: Ch. Ro, M. Cashel and Ll. Fernández-Coll

In this manuscript Ro et al., investigated the relationship between the alarmone ppGpp, the Catabolite Represor Protein CRP and the second messenger cAMP in regulating the expresión of the lacZ gene from Escherichia coli. Employing genetic, physiologic and immunologic approaches, the authors described that i) ppGpp indirectly affects the expression of lacZ by affecting the CRP-cAMP complex, ii) ppGpp regulates the expression lacZ through CRPacetylation, iii) YfiQ is responsible of acetylating CRP in a RelA-dependent manner, and finally, iv) RelA regulates in a positive manner the expression of yfiQ. Based on these results, they concluded that ppGpp induce the acetylation of CRP, thus affecting its regulatory properties

with a direct impact on the expression of the lacZ gene.

Below please find my comments to the author´s responses.

Major comments:

1) Lines 138-139: In the experimental approach the authors determined beta-galactosidase levels to evaluate the transcriptional control exerted by cAMP-CRP over the lacZ operon. In these paragraphs they indicate that addition of IPTG (1 mM) eliminate the repressor effects of LacI; however, in most of the experiments the authors don’t indicate if the minimal media employed with different carbon sources was supplemented with IPTG. Furthermore, with such amenable model of study, it calls the attention that the authors did not employ a lacI-deficient strain to unambiguously rule out the repressor effects of LacI.

As indicated by You et al. 2013, adding 1 mM of IPTG mimics a LacI-deficient background, having no effect on the correlation between lacZ levels and growth rate. Thus we decided to not include it on our manuscript, because we believe that it has been already proven. This is now stressed in the text (lines 147-148).

Reviewer comment (RC): Comment appropriately addressed.

2) Lines 146-51: The authors justify the advantage of using a RelA-deficient strain instead of a double relA spoT mutant. The rational is correct; however, as shown in Figure S1, in culture media supplemented with glycerol and glucose the relA mutant still contain ppGpp. Can the authors clarify if this amount of the alarmone is influencing or not their results. Perhaps, the use of an available relA spoT strain would render more confident experiments in a genetic background fully depleted of ppGpp. Also, Figure S1 do not show if there are statistical differences in the levels of ppGpp between the strains and carbon sources employed, please include this information in the revised manuscript.

As mentioned in line 157, a mutant ΔrelA ΔspoT will not grow in the conditions used for this study, minimal media without amino acids (Xiao, et al. 1991). That forces us, as well as other researchers in our field, to use the closest strain possible to a ppGpp0 strain: in this case a ΔrelA strain. RelA-dependent synthesis of ppGpp responds to starvation of amino acids, but also it will be stimulated by starvation of precursors of amino acids (responding to different carbon sources). Therefore, under the tested conditions, most of the ppGpp is synthesized by RelA, and consequently, a strain ΔrelA shows almost undetectable levels of ppGpp (fig. S1). This is further discussed in the revised manuscript (lines 157-164). Moreover, the statistical differences have been included at the figure S1, as requested by the reviewer.

RC: Comment appropriately addressed.

3) As the authors stated in the introduction, the cAMP-CRP complex controls the expression of several catabolic genes; therefore, to better reinforce the conclusions described in this work, the authors must include experiments with reporters additional to lacZ.

We agree with the reviewer that more reporter genes would strengthen our point, the more the merrier. However, we also need to stress that lacZ has been a staple to report effects over the CRP complex for decades. Transcriptomic studies have shown an overlap between the genes regulated by CRP and ppGpp (Traxler et al. 2006), and we believe that our results suggest that some of these overlapping effects could be the result of CRP acetylation promoted by ppGpp. Therefore, to address this hypothesis, new transcriptomic studies that include ΔackA-pta and ΔyfiQ mutant strains are required. Evidently, this will be part of a follow up manuscript. This is now discussed in the manuscript (line 359-362).

RC: Comment appropriately addressed.

4) Lines 165-168. The manner of presenting the results in Figure 1 are rather confuse. This reviewer and the interested audience would be benefited if the authors present in a more detailed form these results. Please move the results of Figure S2-A to the main text with the following considerations:

A) It is not clear the growth phase in which the strains were analyzed; therefore, these plots must show the growth levels by OD600nm and ideally by viable counts.

B) In the same Figure, plot in a time dependent fashion, the levels of beta-galactosidase of the strains analyzed in the distinct carbon sources employed

The figure 1 is based on the experiments described by You et al. 2013, as indicated on the text, thus we decided to plot it in the same way. There the authors show a correlation between growth rate (μ) and lacZ expression. Then, we believe that plotting OD600nm and β-galactosidase activity in a time-dependent manner may confuse the reader from the main point of the figure, that the β-galactosidase activity is directly proportional to the growth rate and this can be modified by ppGpp. We agree that Figure S2A will help on the comprehension of the figure, and we have moved it to the main text, as suggested by the reviewer. All the samples for measuring β-galactosidase activity were taken at OD600nm of 0.1. Now, this is also added to the figure legend. The growth levels for each strain are indicated with the growth rates (μ) calculated with the OD600nm.

RC: Comment addressed.

C) Here, a question arises: what is the functional relationship between RelA and Crp in regulating LacZ levels in a LacI-deficient background? This question can be easily tested in relA crp genetic background but plotting the results in a time dependent manner.

This is an interesting point raised by the reviewer. As previously discussed, You et al. 2013 show that the addition of IPTG mimics a LacI-background, and does not affect the regulation of LacZ levels. So, we consider that addition of 1 mM of IPTG has a similar effect as a LacIdeficient background. As mentioned above, this is now stressed in the manuscript. We also agree with the reviewer that a classic way to determine the involvement of CRP on the regulation of ppGpp on lacZ expression, it would be the use of a Δcrp mutant strain. However, a Δcrp strain does not express lacZ (undetectable β-galactosidase activity) and it does not grow in minimal media supplemented with glycerol. Therefore, we decided to include the experiments using the lacUV5 promoter (that do not contain the CRP-binding site), despite it is just a confirmation of a previous result (Yang et al. 1974).

RC: Ok

5. Lines 169-176:

5.1. Lines 169-170. Please be more specific in explaining how the authors expect that ppGpp could affect the lacZ promoter; more specifically, is not expected that ppGpp regulates lacZ (RFP in their experiments) expression by binding RNA polymerase? Please see https://doi.org/10.1073/pnas.1819682116

It has previously shown (Yang et al. 1974) that lacUV5 does not respond to the levels of ppGpp. Actually, it has been used by several authors as negative control in experiments of in vitro transcription when studying ppGpp regulation. Then, the experiments in figure S2 need to be taken as a verification of a previous result. We have stressed this notion in the reviewed version of the manuscript (line 191). As pointed by the reviewer, genes directly regulated by ppGpp show a GC-rich discriminator (region between -10 box and +1) if repressed by ppGpp or an Atrich discriminator if stimulated. Neither lacUV5 nor lac promoter show such discriminator. This information is now added to the manuscript (lines 185-188).

RC: Ok

5.2. In the results of Figure S2-B, please make sure to explain:

A) that you are using a PlacUV5-Rfp fusion in an extrachromosomal multi-copy plasmid

B) the experiment does not directly measure lacZ, but RFP expression.

This has been mentioned in the modified version of the manuscript (line 191), and the figure legend.

RC: Ok

C) with a single experimental time-point is difficult to verify that ppGpp (RelA deficiency) does not affect lac-RFP expression, these experiments must be performed in time-dependent fashion plotting also the growth curve, ideally as indicated in the next comment.

We agree with the reviewer that this kind of experiments require more than one time-point, and we apologize for the lack of clarity in the figure. In fact, in figure S2 we are not representing the fluorescence of a single time-point, but the rate of production per OD600nm (slope obtained after plotting fluorescence accumulation vs. OD600nm). This is now mentioned in the modified version. However, considering that this experiment is the verification of something previously described (Yang et al. 1974), we do not think that it is necessary to add extra plots or the growth curve.

RC: Ok

D) to avoid effects of gene dosage and more appropriately address these experiments, the authors must have considered to work with a strain carrying a single chromosomal copy of the PlacUV5-lacZ fusion.

As previously mentioned, this was a confirmation of a published result (Yang et al. 1974). Thus, we believe that it was well established in the original paper.

RC: Ok

6. Lines 178-184. Here, the authors stated that the absence of RelA has no effect on the levels of cAMP-CRP. To support this notion, they, independently, analyzed protein levels of CRP, cAMP concentrations and mRNA levels of cyaA. However, to better support this conclusion the authors are strongly encouraged to determine the levels of the cAMP-CRP complex.

We agree with the reviewer that we determine the effect of ppGpp over the levels of CRP and cAMP but not the levels of the cAMP-CRP complex. If we mentioned on the text that “ppGpp do not affect the cAMP-CRP complex” it has been for a matter of simplicity. This has been modified in the text accordingly. About the recommendation to measure the levels of the complex, first, we do not have the capacity or the experience to perform such experiment. And second, there is no evidence to suggest that ppGpp can physically interfere with the binding of cAMP to CRP. Despite that ppGpp can bind to some proteins, most of the effects are through changing gene expression. Few protein interactions with ppGpp have been described lately, but none with CRP. There are no evidences either that ppGpp can directly interact with cAMP. It is arguable that the acetylation of CRP could interfere with the formation of the complex with cAMP, but as discussed in the text (lines 317-318), that will have to be determined in future studies.

RC: Ok

7. CRP acetylation experiments (Fig. 2; Figs. S5, S6 and Table 1)

A) Figure 2. Notation of strains tested in Fig. 2 is confuse, please clearly indicate the genotype of each strain and present the results in the following order: WT, ackA-pta, relA, relA/ackA-pta establishing significant differences among the four strains. Please, describe and interprete these comparisons in the main text of the paper.

The aim of the figure 2 is to show that the effect of ppGpp over lacZ requires the acetylation of CRP. First, we have to clarify that panel A and B show β-galactosidase activity experiments, while panel C show CRP’s acetylation levels only in the presence or the absence of RelA. This is now stressed in the figure legend.

Second, the nomenclature used in this figure is the same as used in the rest of the manuscript to ensure the cohesion of the manuscript. In panel A and B, the presence of RelA is indicated by color (as indicated at the figure legend). Any other genetic background is indicated in the Xaxis. In panel C, it is true that the color code is broken (both columns are black), but there is only one category. Third, the acetylation of CRP in ΔackA-pta and ΔyfiQ mutant strains is shown only in Table1 (discussed below).

RC: Ok

B). Figs S5 and S6 and Table 1. With the immunological (Western blotting) approach employed to determine levels of acetylated CRP in the distinct genetic backgrounds, is a little difficult to agree with the conclusion of the authors that relA, ackA-pta, yfiQ and cobB regulate the levels of acetylated CRP. Even in the absence of RelA and YfiQ or RelA and AckA-Pta, the anti Ac-K antobodie detects bands of similar intensity than those observed in the WT strain. Furthermore, the protein band control (which I assume is RpoA) shows different intensities. Importantly, a significant fraction of acetylated CRP is detected in all the genetic strains analyzed making more difficult to interprete these results. Therefore, the authors are strongly encouraged to find alternative strategies to quantitate levels of acetylated CRP for instance by LC–MS/MS analysis.

Figures S6 and S7 contain the Western blots to determine the levels of acetylated CRP shown in Figure 2C and Table 1, respectively. We agree that the anti Ac-K antibody detects bands that, at naked eye, seem to be of similar intensity but, those intensities were normalized to the amount of CRP detected at the same samples. Moreover, the intensities are not measured de visu but through the software Image Studio from Li-Cor. This information is now added to the manuscript (line 113-114). Once the detected intensities were normalized to the levels of CRP, the amount of acetylated CRP in a ΔrelA strain happen to be 2-fold lower than WT, and this is reported in fig. 2C. As we interpret this result, ppGpp seems to stimulate the acetylation of CRP under the conditions tested. In figure S7, showing the Western blots used for the table 1, we are determining the effects of YfiQ and AckA-Pta over the levels of CRP (normalized to RpoA) and the levels of acetylated CRP (normalized to the levels of CRP). Here, we observe that the measured intensities of acetylated CRP (again, normalized to CRP) in a ΔyfiQ background are 2-fold lower than WT, but independent of the presence or the absence of RelA. We interpret these results as the effect of ppGpp over CRP acetylation seems to require the presence of YfiQ, and this is how it is now stated on the text.

Despite that LC-MS/MS will give us quantitative levels of acetylated CRP, the antibody used in this manuscript have been used to obtain relative amounts of acetylated proteins in manuscript previously published by our research group, and other research groups in our field. We have stressed that our results are relative amounts instead of quantitative levels (line 247).

RC: Experiments shown in Figs S6 and S7 are relevamt to support a major conclusion of the manuscript, therefore they must be shown in the main text.

8. The conclusion that YfiQ is responsible of acetylating CRP must be clearly documented as this is a key evidence of the regulation mechanism of CRP proposed by the authors. Please specifiy if CRP has been described as a target for YfIQ. If not, the authors are encouraged to implement a more specific approach to demonstrate this fact, for example, using in in vitro assays the unacetylated form of pure CRP as a substrate for the purified form of YfiQ.

Acetylation of CRP, yfiQ-dependent, have been previously documented by Kuhn et al. 2014. This has been further discussed, as requested by reviewer 1 (as can be seen above, lines 314- 317). It is true that further studies are required to determine more in detail what residues are acetylatilated in a ppGpp-dependent manner, and how does it impair lacZ transcription. But, as it is now discussed in the modified text (lines 317-318), this will be the focus of future manuscripts.

RC: Ok

9. Finally, regarding the conclusion that yfiQ expression is regulated by the levels of ppGpp (Fig. 3), the authors must indicate in the discussion section how the alarmone may exert its regulatory functions.

We absolutely agree with the reviewer. As previously said, genes directly stimulated by ppGpp has an A/T rich discriminator. As shown by Castaño-Cerezo et al. 2011, the expression of yfiQ is stimulated in stationary phase, but it does not respond to the alternative sigma factor RpoS, suggesting that any effect must be direct. This is now discussed at the modified version of the manuscript (lines 297-299).

RC: Ok

Minor comments.

1) Lines 98-101: Please describe in more detail this protocol, indicate the substrate used and specify how the Miller Units were determined.

This protocol is a well established standard in our field, but, as suggested by the reviewer, more information has been added.

RC: Comment appropriately addressed.

2) Lines 123-126. Please show as a supplementary Figure, a typical growth curve to see the diauxic effect and clearly indicate in this curve the OD600nm at which most of the sample were taken for biochemical and immunological determinations.

We agree that adding a diauxic growth curve will help to understand some parts of the manuscript; it is now a panel of figure S5. However, we believe that indicating there the OD at which most of the samples were taken will be misleading for the reader, considering that only the experiment shown in figure S5 was made during diauxic shift. Instead, we have stressed in all the figure legends that the samples were taken during exponential phase (at an OD600nm of 0.1).

RC: Comment appropriately addressed.

3) Figs. S3 and S7. Please describe in the Figure Legends the purpose of the Western Blotting of RpoA in these experiments.

This is now added to the figure legends as sugg

**********

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Reviewer #1: No

Reviewer #2: No

Acceptance letter

Benjamin J Koestler

19 Oct 2021

PONE-D-21-19172R1

The secondary messenger ppGpp interferes with cAMP-CRP regulon by promoting CRP acetylation in Escherichia coli.

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Levels of ppGpp.

    The amount of ppGpp was measured relative to total amount of G (pppGpp + ppGpp + GTP) by TLC in MG1655 and ΔrelA strains grown in MOPS media with 0.2% glucose or 0.4% glycerol. Error bars represent SD of two biological replicates. Statistical significance was measured with T-student test (*p-value < 0.05, n.s. p-value > 0.05).

    (TIFF)

    Click here for additional data file. (461.6KB, tiff)
    S2 Fig. The effect of ppGpp on lacZ expression requires of CRP binding site.

    MG1655 (WT) and CF18005 (ΔrelA) harboring the plasmid pBbA5k were grown in M9 minimal media supplemented with glycerol 0.4% and IPTG 1 mM. Finally, fluorescence emitted by RFP (Red Fluorescent Protein) was measured. The amount of fluorescence was plotted against the OD600, giving a linear correlation where the slope is the RFP production rate per OD600. Error bars represent SD. Statistical significance was measured with T-student test (n.s. p-value > 0.05).

    (TIFF)

    Click here for additional data file. (378.7KB, tiff)
    S3 Fig. ppGpp does not affect levels of CRP or cAMP.

    (A) MG1655 (WT) and CF18005 (ΔrelA) were grown in M9 minimal media supplemented with either glucose 0.2% or glycerol 0.4% up to exponential phase (OD600 0.1) and protein levels of CRP and RpoA were measured by Western blot. Means and standard deviation are shown of CRP amounts normalized to the amounts of RpoA (loading control) relative to the values from WT in glucose. (B) Measurements of cAMP of cells grown as in panel (A). Error bars show standard deviation of 2 biological samples. Statistical significance was measured with T-student test (n.s. p-value > 0.05).

    (TIF)

    Click here for additional data file. (3.9MB, tif)
    S4 Fig. The gene expression of cyaA measured by qPCR from WT and ΔrelA in minimal media with glucose or glycerol.

    Expression levels are normalized to the expression in WT grown in glucose. Error bars show standard deviation of 2 biological samples and 3 technical replicates. Statistical significance was measured with T-student test and no difference was observed between WT and ΔrelA (p-value > 0.05).

    (TIF)

    Click here for additional data file. (363.6KB, tif)
    S5 Fig. The KAT YfiQ does not affect the diauxic shift.

    (a) The strain MG1655 (wt) and ΔrelA, together with their isogenic ΔyfiQ and ΔcobB mutants were grown in M9 with 0.025% glucose and 0.4% lactose for 12 h and OD600 measured every 10 min. Ratios of diauxic time normalized to generation times of three independent experiments with duplicate wells (six values) were plotted as box plots. Bottom and top of the colored box represent first and third quartiles, and the band inside the box is the median. Whiskers represent minimum and maximum data, while circles are outliers (single points). (b) Typical diauxic growth curve from WT strain.

    (TIFF)

    Click here for additional data file. (1.6MB, tiff)
    S6 Fig. ppGpp controls acylation of CRP, Westen blots.

    The amount of acylated CRP was measured by Western blot from total extracts of WT and ΔrelA strains together with their isogenic ΔcobB mutants, were grown in M9 minimal media supplemented with glycerol 0.4% up to OD600 of 0.1. To ensure a proper identification of the CRP protein, a Δcrp mutant was also added. The amount of acetylated protein (detected by Western blot using antibody specific for acetylated lysines, Ac-K) was normalized to the amount of CRP and presented relative to the WT strain. STDV = standard deviation.

    (TIF)

    Click here for additional data file. (4.3MB, tif)
    S7 Fig. YfiQ acetylates CRP, Western blots.

    The amount of acylated CRP was measured by Western blot from total extracts of the WT strain, together with the ΔackA-pta, ΔackA-pta ΔrelA, ΔyfiQ and ΔyfiQ ΔrelA strains. Cells were grown in M9 minimal media supplemented with glycerol 0.4% up to exponential phase (OD600 0.1). To ensure a proper identification of the CRP protein, a Δcrp mutant was also added. The amount of acetylated protein (detected by Western blot using antibody specific for acetylated lysines, Ac-K) was normalized to the amount of CRP and presented relative to the WT strain. The CRP amounts are normalized to the amounts of RpoA (loading control).

    (TIF)

    Click here for additional data file. (3.8MB, tif)
    S1 Table. Bacterial strains and plasmids used in this study.

    (PDF)

    Click here for additional data file. (257KB, pdf)
    S2 Table. List of primers used in this report.

    (PDF)

    Click here for additional data file. (133.5KB, pdf)
    S1 File. Uncropped and unadjusted Western blot images.

    (PDF)

    Click here for additional data file. (563.5KB, pdf)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (36.4KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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