Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation

Simon Diez; Molly Hydorn; Abigail Whalen; Jonathan Dworkin

doi:10.1371/journal.pgen.1009957

. 2022 May 20;18(5):e1009957. doi: 10.1371/journal.pgen.1009957

Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation

Simon Diez ¹, Molly Hydorn ¹, Abigail Whalen ¹, Jonathan Dworkin ^1,^*

Editor: Sean Crosson²

PMCID: PMC9173625 PMID: 35594298

Abstract

Phenotypic heterogeneity of microbial populations can facilitate survival in dynamic environments by generating sub-populations of cells that may have differential fitness in a future environment. Bacillus subtilis cultures experiencing nutrient limitation contain distinct sub-populations of cells exhibiting either comparatively high or low protein synthesis activity. This heterogeneity requires the production of phosphorylated guanosine nucleotides (pp)pGpp by three synthases: SasA, SasB, and RelA. Here we show that these enzymes differentially affect this bimodality: RelA and SasB are necessary to generate the sub-population of cells exhibiting low protein synthesis whereas SasA is necessary to generate cells exhibiting comparatively higher protein synthesis. Previously, it was reported that a RelA product allosterically activates SasB and we find that a SasA product competitively inhibits this activation. Finally, we provide in vivo evidence that this antagonistic interaction mediates the observed heterogeneity in protein synthesis. This work therefore identifies the mechanism underlying phenotypic heterogeneity in protein synthesis.

Author summary

Upon encountering conditions unfavorable to growth such as nutrient limitation, bacteria enter a quiescent phenotype that is mediated by group of guanosine nucleotides collectively known as (pp)pGpp. These nucleotides direct the down-regulation of energy intensive processes and are essential for a striking heterogeneity in protein synthesis observed during exit from rapid growth. Here, we show that a network of (pp)pGpp synthases is responsible for this heterogeneity and describe a mechanism that allows for the integration of multiple signals into the decision to down regulate the most energy intensive process in a cell.

Introduction

Nutrient availability is a major environmental cue for bacteria. For example, amino acid starvation results in induction of the stringent response, a conserved mechanism dependent on the synthesis of the nucleotides guanosine penta- and tetra-phosphate ((p)ppGpp). These nucleotides mediate a broad shut down of energy intensive reactions which are required during rapid growth [1,2]. (p)ppGpp directly binds and inhibits key proteins that catalyze processes including transcription (RNA polymerase [3,4]), translation (GTPase IF2 [5]), GTP biosynthesis (HprT and GmK [6]), DNA replication (DNA primase [7]), and ribosome assembly (ObgE [8]).

Gram-positive bacteria typically encode a single, bi-functional RSH (RelA-SpoT Homolog) enzyme capable of both (p)ppGpp synthesis and hydrolysis as well as two additional small alarmone synthases (SAS) that lack hydrolytic activity. Unlike RSH proteins, which are activated by the binding of a deacylated tRNA in the A-site of the ribosome, SAS enzymes are often transcriptionally regulated [9] and some are also under allosteric control [10]. RelA/SpoT and the SAS synthases preferentially produce different molecules in different species. For example, in response to amino acid starvation, E. coli RelA produces approximately equal amounts of the tetra-phosphorylated (ppGpp) and the penta-phosphorylated (pppGpp) guanosines, whereas B. subtilis RelA primarily generates pppGpp using GTP and ATP as substrates [11]. B. subtilis SasB preferentially utilizes GDP and ATP to generate the tetra-phosphorylated guanosine (ppGpp) [12]. SasA, the other SAS enzyme in B. subtilis, generates either a 5’ monophosphate 3’ di-phosphate guanosine or a 5’ di-phosphate 3’ monophosphate (pGpp) [12], at least in part due to the action of the NahA hydrolase [13]. Together, these three closely related nucleotides are referred to as (pp)pGpp.

Recently, our laboratory demonstrated that accumulation of (pp)pGpp attenuates protein synthesis when populations of B. subtilis cease growing exponentially [5]. This attenuation is bimodal and results in a heterogeneity in the protein synthesis activity of individual cells that exhibit either comparatively high or low protein synthesis activity [5]. Here we find that all three B. subtilis (pp)pGpp synthases including the RSH protein RelA and SAS proteins SasA and SasB are required for this heterogeneity since the absence of any of these synthases results in the loss of bimodality. The SasA product pGpp and the RelA product pppGpp together antagonistically regulate activation of the third synthase (SasB), that is itself responsible for the synthesis of ppGpp, a molecule that directly inhibits the initiation of translation during nutrient limitation [5].

Results

The SasA and SasB (pp)pGpp synthases contribute to heterogeneity

Cellular heterogeneity in protein synthesis as B. subtilis cultures exit rapid growth is dependent on the presence of the phosphorylated guanosine nucleotides (pp)pGpp [5]. We investigated the origins of this heterogeneity by assessing single cell protein synthesis using O-propargyl-puromycin (OPP) incorporation in strains carrying deletion mutations in either of the two B. subtilis (pp)pGpp synthases (SasA and SasB) whose expression increases during exit from rapid growth [12]. To quantify these effects we applied a cutoff that specifies the population of cells with low rates of protein synthesis. Nearly all cells of a B. subtilis stationary phase culture exhibit very low protein synthesis [5] so we defined this cutoff (850 arbitrary fluorescence units (au)) as the magnitude of OPP labeling of a B. subtilis culture in stationary phase that captures >95% of the entire population (S1 Fig). We used this threshold to define the fraction of the population with low rates of protein synthesis during late transition phase (OD₆₀₀ ~0.685) as “OFF” (S2 Fig). By convention, we define the remainder of the population as “ON.”

A strain lacking SasB (ΔsasB) contained fewer “OFF” cells as compared to the wildtype strain (Fig 1A and 1B). This result is consistent with our previous observation that the SasB product ppGpp inhibits the function of IF2 and thereby downregulates protein synthesis [5]. In striking contrast, a strain lacking SasA (ΔsasA) did not contain the substantial fraction of “ON” cells seen in the wildtype parent strain (Fig 1A and 1C) and most cells in the population were “OFF”. This observation suggests that the SasA product pGpp does not inhibit translation, as does the SasB product ppGpp. Consistently, unlike ppGpp, pGpp does not directly bind known translational GTPases (e.g., EF-G [14])

sasA but not sasB expression is correlated with levels of protein synthesis

sasA and sasB are regulated transcriptionally and expressed post-exponentially [12,15] when the heterogeneity is observed (Fig 1A). We therefore asked if expression of either sasA or sasB is correlated with protein synthesis using transcriptional fusions of the sasA or the sasB promoters to the gene encoding YFP (P_sasA-yfp or P_sasB-yfp). Consistent with prior observations [12], expression of both sasA and sasB reporters increased during the exit from exponential growth (Fig 2A and 2B). We examined the relationship between promoter activity and protein synthesis by measuring both YFP expression and OPP incorporation in single cells. Cells with higher sasA expression (P_sasA-yfp) were more likely to have higher levels of protein synthesis than cells with lower sasA expression (Fig 2D). If the population is divided into quartiles of sasA expression, average OPP incorporation in the top two quartiles as compared to the bottom quartile was significantly higher (Fig 2D). In comparison, the difference in OPP incorporation between any of the quartiles of sasB expression (Fig 2C) was not significant. Thus, differences in sasA, but not sasB, expression are associated with the observed heterogeneity in protein synthesis.

SasB allosteric activation is necessary for heterogeneity

If changes in sasB transcription are not associated with differences in protein synthesis (Fig 2C), but SasB is necessary for the heterogeneity of protein synthesis (Fig 1B), what mechanism underlies differential SasB activity in single cells? B. subtilis SasB is subject to allosteric activation by pppGpp, the main product of B. subtilis RelA [16]. Phe-42 is a key residue in this activation and a SasB mutant protein carrying an F42A substitution (SasB^F42) is not allosterically activated by pppGpp in vitro [16]. We investigated the importance of this allosteric activation for protein synthesis heterogeneity using a strain expressing SasB^F42 instead of SasB. Since heterogeneity of this strain was significantly attenuated compared to the WT strain (Fig 3A and 3B), allosteric activation of SasB by pppGpp is key for the bimodality of protein synthesis activity.

Fig 3 — **(A, B,C)** Representative pictures and population distributions of OPP labeled **(A)** wildtype (JDB1772), **(B)** *sasB*^F42A (JDB4340), and **(C)** *relA*^Y308A (JDB4300) strains during late transition phase. % of population “OFF” as determined in S1 Fig is presented above each representative distribution. Statistical significance was determined by comparing 3 independent populations of WT to either mutant. P-values are 0.002 and 0.035 for *sasB*^F42A and *relA*^Y308A respectively.

This result suggests that the enzyme responsible for pppGpp synthesis could also affect the heterogeneity. RelA is the primary source of pppGpp in B. subtilis [11], so loss of relA would be predicted to affect SasB activity. We therefore generated a strain expressing a RelA mutant protein (RelA^Y308A) carrying a single amino acid change at a conserved residue essential for synthase but not hydrolysis activity [17,18] since RelA hydrolytic activity is essential in a strain that retains functional sasA and sasB genes [19]. Labeling of this strain with OPP in late transition phase revealed that the “OFF” population was largely absent (Fig 3C), demonstrating that RelA-mediated pppGpp synthesis is important for the bimodality.

SasB allosteric activation is inhibited by pGpp

A strain lacking SasA (ΔsasA) contains more “OFF” cells as compared to the wildtype parent (Fig 1C). The presence of this sub-population of cells depends on a SasB protein that can be allosterically activated (Fig 3B). Integrating these two observations, we hypothesized that a product of SasA (pGpp) inhibits the allosteric activation of SasB by pppGpp. pGpp and pppGpp could have an antagonistic interaction since they are likely capable of binding to the same site on SasB, but their differing phosphorylation states could affect their ability to allosterically activate SasB.

We tested this possibility by assaying in vitro whether pGpp inhibits the allosteric activation of SasB. First, we confirmed that SasB generates more ppGpp when reactions are supplemented with pppGpp and, as reported [16], we observed a ~2 fold increase in ppGpp production when SasB was incubated with pppGpp (Fig 4A). Using pGpp synthesized in vitro by the (pp)pGpp hydrolase NahA [14], we observed that pGpp attenuates the allosteric activation of SasB in a dose dependent manner (Fig 4A). Since even the highest concentration of pGpp did not decrease production of ppGpp relative to that generated by SasB without the addition of pppGpp (Fig 4A), the inhibition is likely specific to the allosteric activation. We tested this directly by assaying the effect of pGpp on SasB activity in the absence of its allosteric activator (pppGpp). Addition of pGpp did not significantly affect SasB activity within the range of pGpp concentrations we used previously (S3 Fig). We further confirmed the specificity by assaying a SasB^F42 mutant protein that is insensitive to allosteric activation by pppGpp [16]. As reported SasB^F42A has similar activity to a non-allosterically activated WT SasB in the presence of pppGpp (Fig 4B). However, in contrast with wildtype SasB, pGpp does not affect the activity of SasB^F42A even when pppGpp is included (Fig 4B).

Fig 4 — **(A)** Representative TLC of nucleotides present following incubation of wildtype SasB with [α-³²P]-ATP and GDP in the presence or absence of pppGpp and increasing concentrations of pGpp (μM) (top). Quantitation of the ratio of ppGpp to total nucleotides present in each lane in TLC. This ratio was calculated using the formula: ppGpp/ATP + ppGpp (bottom). **(B)** Representative TLC of nucleotides present following incubation of SasB^F42A with [α-³²P]-ATP and GDP in the presence or absence of pppGpp and increasing concentrations of pGpp (top). Ratio of ppGpp present in each lane in TLC as determined the formula, ppGpp/ATP + ppGpp (bottom). Statistical analysis (two tailed t-test) showed no significance (p>0.05) between reactions containing SasB in the presence or absence of pppGpp and/or pGpp.

These in vitro biochemical experiments suggest that the effect of SasA on protein synthesis heterogeneity is dependent on the activity of SasB. If this is true in vivo, then a ΔsasB mutation should be dominant to a ΔsasA mutation. Consistently, the population of "OFF" cells in a ΔsasA strain was absent in a strain lacking both SasA and SasB (ΔsasA ΔsasB) (Fig 5A and 5B). Thus, the effect of SasA is dependent in vivo on SasB. Finally, since RelA activates SasB, a relA mutation should be dominant to a ΔsasA mutation. Consistently, a strain expressing RelA^Y308A and carrying a ΔsasA mutation exhibited a loss of heterogeneity in protein synthesis similar to the relA^Y308A strain, demonstrating that the effect of the ΔsasA mutation depends on a functional RelA synthase (Fig 5A and 5C). Since pGpp also accumulates in stationary phase cells as a result of degradation of both ppGpp and pppGpp by the hydrolase NahA [14,20], we asked if NahA contributes to the heterogeneity in protein synthesis by comparing OPP incorporation in wildtype and ΔnahA cells during late transition phase. Since we observed no difference in heterogeneity (S4 Fig), SasA is the primary regulator of heterogeneity under our experimental conditions.

Fig 5 — **(A, B, C)** Representative pictures and population distributions of OPP labeled **(A)** *ΔsasA* (JDB4310), **(B)** *ΔsasA ΔsasB* (JDB4312) **(C)** Δ*sasA relA*^Y308A (JDB 4301) strains during late transition phase. % of population “OFF” as defined in S1 Fig is presented above each representative distribution. Statistical significance was determined by comparing 3 independent populations of WT to either mutant. P-values are 0.028 and 0.030 for *ΔsasAΔsasB* and *ΔsasA relA*^Y308A, respectively.

Discussion

B. subtilis populations experiencing nutrient limitation and entering into quiescence respond bimodally with respect to global protein synthesis activity [5]. Here, we find that this bimodality depends on all three (pp)pGpp synthases. We demonstrate that it is dependent on the allosteric activation of SasB by the RelA product pppGpp and that this activation is antagonized by a SasA product pGpp. Our work therefore provides a mechanism for the phenotypic heterogeneity observed and identifies a novel regulatory interaction between (pp)pGpp synthases.

Regulation of protein synthesis during nutrient limitation

The down-regulation of protein synthesis in B. subtilis cells experiencing nutrient limitation occurs in part as a result of ppGpp directly inhibiting IF2 [5]. SasB is the main source of ppGpp and this work identifies how ppGpp synthesis by SasB and the subsequent downregulation of protein synthesis is coupled to changes in environmental conditions. First, SasB allosteric activation by the RelA product pppGpp is required for the downregulation of protein synthesis in a subpopulation of cells (Figs 3 and 5). RelA activity reflects tRNA charging levels [11], thereby coupling SasB-dependent regulation of protein synthesis to amino acid availability. Second, a SasA product (pGpp) inhibits the allosteric activation of SasB (Fig 4). Although SasA is constitutively active, sasA expression, at least in part, reflects availability of the Lipid II peptidoglycan precursor [21–23], thereby coupling SasB-dependent regulation of protein synthesis to cell wall metabolism. Thus, RelA and SasA dependent regulation of SasB may integrate multiple environmental signals in the decision to attenuate protein synthesis.

Physiological sources of variability in SasB activity

Phenotypic heterogeneity such as that observed here in the context of protein synthesis can arise from stochastic differences in gene expression [24]. Although sasB expression exhibits substantial variability in expression cell to cell (Fig 2A), it does not correspond with the level of protein synthesis in individual cells (Fig 2C). Thus, variability of SasB activity in single cells is likely relevant. What could be responsible? Our observations link heterogeneity to the convergent regulation of SasB allosteric activation by the products of the RelA and SasA synthases (Fig 4A). Thus, both enzymes are potential sources of variability and, consistently, strains carrying either relA^Y308A or ΔsasA mutations exhibit a loss in heterogeneity as compared to the wildtype (Figs 1C and 3C). Since RelA is a cellular sensor of tRNA charging, levels of which are highly sensitive to growth conditions [25], variations in this parameter could contribute to variability in protein synthesis via modulation of RelA activity. Noise in sasA transcription is dependent on the activity of PrkC, a membrane Ser/Thr kinase that regulates sasA via the essential WalRK two component system [21]. Since both WalRK [26] and PrkC [27] activities reflect cell wall metabolism, variation in this process could also impact sasA variability. Thus, differences in the protein synthesis activity of individual cells may reflect cellular variations in amino acid and/or cell wall metabolism.

Allosteric activation of (pp)pGpp synthases

Many genes encoding SAS proteins such as sasB are transcriptionally regulated [9]. In addition, we observe here that allosteric activation of SasB by pppGpp [16] is required for the attenuation of protein synthesis (Fig 3) demonstrating that sasB transcription is necessary but not sufficient, at least in the physiological context of nutrient limitation. We also find that this allosteric activation is antagonized by a SasA product pGpp, consistent with the epistatic relationship between sasB and sasA (Fig 5A). Antagonistic regulatory mechanisms are likely widespread in this family of synthases. For example, the SasB homolog Enterococcus faecalis RelQ is attenuated by RNA that competes with pppGpp for binding to the allosteric site [28]. Given the very recently observed allosteric activation of B. subtilis RelA by (p)ppGpp [29], an important question for future study is to determine whether this activation is also subject to antagonism by pGpp and, if so, to characterize the physiological consequences of this regulation.

(pp)pGpp synthases

Strains carrying single mutations in one of the three genes encoding a (pp)pGpp synthase (ΔsasA, ΔsasB, relA; Figs 1 and 3C) exhibit different protein synthesis activity, consistent with previous reports that SAS enzymes differ between themselves and also with RelA in the guanosine nucleotide that they preferentially produce [14,30–32]. Our experiments thereby extend previous observations that ppGpp and pppGpp can differ in their effect on gene transcription in E. coli [33]. The biochemical experiments demonstrating that pGpp antagonizes pppGpp allosteric activation of SasB, but itself is not capable of activation (Figs 4A and S3) are consistent with our physiological experiments. The biochemical activity of these nucleotides have been reported to differ, including observations that pppGpp is much more potent than ppGpp in stimulating SasB [16], that pGpp is a significantly more potent inhibitor of purine salvage enzyme XPRT than ppGpp [34], and that ppGpp, but not pppGpp, inhibits the function of IF2 in stimulating subunit joining [35].

Physiological implications of heterogeneity in protein synthesis

(pp)pGpp has long been thought to mediate entry into bacterial quiescence [36,37]. This transition facilitates survival in nutrient limited environments and its regulation depends upon the integration of a multitude of rapidly changing environmental signals that themselves may impair decision-making. One way bacteria deal with such uncertainty is to generate subpopulations, with distinct, often bimodal phenotypes from a population of genetically identical cells [24]. Examples of phenotypic variation in B. subtilis include heterogeneity in specific metabolic activities such as acetate production [38] or in developmental transitions such as sporulation [39] and competence [40]. The phenotypic variation in protein synthesis activity we observe here has potentially important functional implications. A global reduction in protein synthesis activity, if accompanied by a constant rate of protein degradation, would have the effect of reducing overall metabolic capacity, especially by affecting processes like ribosome assembly. Global effects also could have specific regulatory consequences. For example, the alternative sigma factor B. subtilis SigD drives expression of genes controlling daughter cell separation and motility that exhibit well characterized phenotypic variation. RelA affects both this variability as well as absolute levels of SigD [41], suggesting that differences in protein synthesis between cells may contribute to SigD variability.

In summary, this work demonstrates that the three (pp)pGpp synthases comprise a signaling network responsible for the heterogenous regulation of protein synthesis as B. subtilis cultures enter quiescence. We find that this heterogeneity is dependent on the RelA product pppGpp, which allosterically activates SasB, and a SasA product, pGpp, which antagonizes this activation. Since the activities of RelA and SasA reflect amino acid and peptidoglycan precursor availability, respectively, these parameters are thereby coupled to protein synthesis activity and facilitate cell decision making during the entry into quiescence.

Materials and methods

Strains and media

Strains were derived from B. subtilis 168 trpC2. sasA (ywaC) and sasB (yjbM) gene knockouts were from transformed into B. subtilis 168 trpC2 using genomic DNA from BD5467 [42]. The sasB transcriptional reporter strain was constructed similarly as described [21]. Briefly, a 107 bp region encompassing the sasB operon promoter (P_sasB) was amplified and inserted into AEC 127 using EcoRI and BamHI sites. The resulting AEC 127 P_sasB was integrated into B. subtilis 168 trpC2 at sacA. sasB^F42A and relA^Y308A strains were generated using integration of pMINIMAD2 derivatives (pMINIMAD2 sasB^F42A and pMINIMAD2 relA^Y308A, respectively). Briefly, sasB was amplified excluding start and stop codons and F42A mutation was introduced using overlap extension PCR. sasB^F42A was inserted into pMINIMAD2 vector using EcoRI and SalI sites. pMINIMAD2 sasB^F42A vector was transformed into B. subtilis 168 trpC2 using a standard transformation protocol. Transformants were selected for erythromycin resistance at 45°C overnight and grown for 8 hours at RT in LB. Cultures were diluted 1:10 in LB and grown overnight. Cultures were plated for single colonies and grown overnight at 45°C. Single colonies were checked for erythromycin sensitivity and sensitive clones were checked for sasB^F42A allele by Sanger sequencing of sasB amplified genomic region. The relA^Y308A strain was generated in a similar way but EcoRI and BamHI sites were used to insert the relA^Y308A gene into pMINIMAD2.

Growth curves

Growth curves were performed in a Tecan Infinite m200 plate reader at 37°C with continuous shaking and OD₆₀₀ measurements were made every five min. Cultures were grown from single colonies from fresh LB plates grown overnight at 37°C. Exponential phase starter cultures (OD₆₀₀ ~ 0.5–1.5) were diluted to OD₆₀₀ = 0.01 and grown in 96-well Nunclon Delta surface clear plates (Thermo Scientific) with 150 μL per well. All growth curves were done in triplicate and media-only wells were used to subtract background absorbance.

OPP labeling

OPP labeling of cells was as described [5]. Exposure times were 30 msec for phase contrast, and 20 msec for mCherry. Fluorescence intensity of ~1570 single cells per experiment was determined using ImageJ. Cells were binned based on fluorescence intensity using 50 a.u. wide bins in all experiments and number of cells in each bin presented as a histogram.

Protein expression and purification

Wildtype and F42A SasB proteins were expressed and purified essentially as described [16]. Wildtype sasB was amplified from B. subtilis 168 trpC2. The F42A mutation was introduced using overlap extension PCR. WT and sasB^F42A PCR products were inserted into pETPHOS expression vector using EcoRI and BamHI sites. pETPHOS WT sasB and pETPHOS sasB^F42A were transformed into E.coli BL21 and proteins were induced with 1 mM IPTG for 2h at OD₆₀₀ ~0.5. Cells were harvested at 4°C and lysed using a Fastprep (MP biomedicals) in 50 mM Tris (pH 8.0), 250 mM NaCl, 5 mM MgCl₂, 2 mM BME, 0.2 mM PMSF, and 10mM imidazole. Lysates were clarified and bound to a Ni-NTA column (Qiagen) for 1h. Columns were washed using 20 mM imidazole. Protein was eluted using 500 mM imidazole, dialyzed into 20mM Tris, 500 mM NaCl, 5mM MgCl₂, 2 mM BME, and 10% glycerol and stored at -20°C. NahA protein was purified in a similar way except that NahA was induced for 1h at 30°C and NahA expressing cells (JDE3138) were lysed, washed, and eluted in 250 mM NaCl instead of 500 mM.

pGpp synthesis

pGpp was synthesized in vitro by purified NahA enzyme as described [14]. Briefly, 10 nM purified B. subtilis NahA was incubated with 30 mM pppGpp (Trilink Biotechnologies) in 40 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl₂ at 37°C for 1 hour. Reactions were monitored for conversion of pppGpp to pGpp using thin layer chromatography on PEI-cellulose plates in 1.5 M KH₂PO (pH 3.6). Nucleotides were visualized using short wave UV light. NahA enzyme was precipitated using ice cold acetone and nucleotides were stored at -20°C.

SasB activity assays and TLC

SasB activity was assayed by measuring the amount of ppGpp generated similar to [5]. Briefly, 0.8 μM purified B. subtilis WT or F42A SasB was incubated with 0.5 μCi of [γ-³²P]-ATP (PerkinElmer) and 50 μM GDP in 20 mM Tris (pH 7.5), 500 mM NaCl, 5 mM MgCl₂, 2mM BME. SasB was allosterically activated using 12.5 μM pppGpp (Trilink Biotechnologies) and pGpp was added as noted. Reactions were performed in a total volume of 10 μL, and each reaction was incubated at 37°C for 1 min before being stopped using 5 μL of ice cold acetone. Conversion of ATP to ppGpp was visualized using thin layer chromatography on PEI-cellulose plates in 1.5 M KH₂PO₄ (pH 3.6). Plates were dried completely at RT and exposed for 5 min on a phosphor storage screen and visualized (GE Typhoon). ATP and ppGpp spot intensities were quantified using ImageJ.

Supporting information

S1 Fig. Determination of “OFF” cells using stationary phase cells.

Threshold for OPP “OFF” cells was determined as the fluorescence value (850 a.u.) that is higher than >95% of cells of OPP labeled wildtype B. subtilis during stationary phase across three independent experiments. (A) Three representative distributions of OPP labeled wildtype B. subtilis. Gray box shows cutoff for cells with low rates of protein synthesis (“OFF”). (B) Quantitation of % of population below the threshold determined as “OFF” in the three experiments in A (means ± SDs).

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S2 Fig. Late transition phase time point and application of “OFF” cutoff to transition phase cells.

(A) Growth curve of wildtype B. subtilis showing point (OD₆₀₀ ~0.685) where cells were labeled with OPP (dashed line). (B) Representative distribution of OPP labeled wildtype B. subtilis. Gray box shows cutoff for cells with low rates of protein synthesis (“OFF”). Threshold (850 a.u.) is the value higher than >95% of cells of wildtype B. subtilis labeled with OPP in stationary phase across three independent experiments. (see S1 Fig).

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S3 Fig. pGpp does not inhibit SasB basal activity.

(Top) representative TLC analysis of wildtype SasB activity in the absence of allosteric activation (no pppGpp added) and with increasing concentrations of pGpp (uM). (Bottom) ratio of ppGpp calculated using the formula, ppGpp/ATP + ppGpp. Statistical analysis (t-test) showed no significance (p > 0.05) between any reaction containing SasB whether or not pGpp was included. Statistical analysis was performed on three separate experiments (means ± SDs).

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S4 Fig. Effect of nahA on heterogeneity.

(A, B) Representative pictures and population distributions of OPP labeled (A) wildtype (JDB1772), (B) ΔnahA (JDB4095) strains during late transition phase.

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S1 Table. Plasmids used in this study.

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S2 Table. Strains used in this study.

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S3 Table. Oligonucleotides used in this study.

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Data Availability

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

Funding Statement

SD was supported in part by the Columbia University Graduate Training Program in Microbiology, Immunology and Infection (NIH, R01 AI106711). JD was supported by NIH R01GM141953, R35GM141953, R21AI156397, and is a Burroughs-Welcome Investigator in the Pathogenesis of Infectious Disease (#1010084). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Genet. doi: 10.1371/journal.pgen.1009957.r001

Decision Letter 0

Sean Crosson, Lotte Søgaard-Andersen

13 Jan 2022

Dear Dr Dworkin,

Thank you very much for submitting your Research Article entitled 'Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but noted some substantial concerns about the current manuscript. Reviewer 1 has raised concerns with attribution of specific Sas, Rel, and Nah activities to the reported phenotypes. This reviewer notes that providing experimental enzyme activity data would greatly strengthen the presented model. In the absence of such data, the authors’ model would need to be revised. Along this same line, Reviewer 2 raises concerns about evidence for the presented model and attribution of specific sas activities and alarmone levels to the observed phenotypes. Reviewer 1 also raises questions about the determination of “OFF” cells that are important to address.

Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

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PLOS Genetics

Lotte Søgaard-Andersen

Section Editor: Prokaryotic Genetics

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The paper by Diez et al that describes a possible basis for bimodal heterogeneity in translation levels in Bacillus subtilis cells in stationary phase. I have previously reviewed this manuscript for another journal and was disappointed to learn that few, if any, of the suggestions made by either reviewer were incorporated into this version of the manuscript. I thought these comments were generally constructive and have reiterated the key points from both referees here, because they remain fully pertinent.

One key issue is the oversimplification of the model whereby RelA produces pppGpp, SasA produces pGpp and SasB produces ppGpp. Each of these enzymes produces a mixture of products and different authors disagree on what the primary functions are in B. subtilis and closely related organisms. RelA and SasB have both been reported to produce mixtures of pppGpp and ppGpp (PMID: 6111556 ; 26124242). Both SasA and SasB have been reported to produce pGpp (PMID: 26124242 ; PMID: 22950019). Indeed, the primary reference cited in favor of the model that SasA produces pGpp and SasB produces ppGpp (ref 12; PMID: 22950019), was unable to distinguish between pGpp and ppGp by mass-spectrometery and favored the later as the most likely product of SasA in the discussion. This paper was not nearly as categorical as the current authors about which enzyme preferentially produces which product. Lastly, others have shown have shown that although SasA-expressing cells accumulate pGpp, most pGpp is from the conversion of ppGpp to pGpp by the hydrolase NahA (PMID: 32983059 ; 33097692). Ideally the authors should compare the activity of the 3 enzymes side-by-side with GTP, GDP and GMP as substrates. In the absence of such an experiment, the authors should be much more broad-minded about presentation of the model

The fluorescence cut-off value used to define cells as 'OFF' for translation was determined as the magnitude of OPP labeling of a stationary phase culture that captures 95% of the population (850 RFU). Unless I am misinterpreting Figure S1, the box showing the 'OFF' population represents <50% of the cells shown in this experiment, not 95% (I cut out the printed graph and weighed the peaks just to be sure). It also seems strange to assume that 95% of WT cells should be 'OFF' in stationary phase, when the study is all about population heterogeneity after all. Is this based on some previous determination? From visual inspection of the profiles shown throughout the paper, it seems that there is always a valley around 700-750 AU ; this might be a better choice for the cut-off.

Why were quartiles chosen for the analysis in Fig. 2C and D? Wouldn't it have been more logical just to divide into two groups, representing the two peaks? Is quartile 2 significantly different from 3 and 4? If the authors insist onf sticking with quartiles, please show where these fall on the graph using dotted lines. Also, please change the colors in panel 2B. The two shades of green are barely distinguishable.

Supplementary Figures S2, S3, and S5 should be deleted and the % 'OFF' cells +/- error and asterisks for significant differences be reported directly on the corresponding main figures in the text, to avoid having to go looking for this data in the supplementary section.

Please replace the term 'Ratio of ppGpp' on the Y-axis to ppGpp/(ppGpp+ATP).

Both referees had a problem with the use of the term epistatic towards the end of the results section. For me, sasB is epistatic to sasA and relA is epistatic to sasA, because the sasB phenotype dominates in the double sasAB mutant and the relA308 phenotype dominates in the sasA relA308 double mutant, ie cells are mostly 'ON' in both cases.

Minor points:

L56 Define RSH enzyme

L98 I am not sure how the authors arrive at the conclusion that 'This observation suggests that the SasA product pGpp does not directly inhibit translation as does ppGpp, but rather acts indirectly'. If anything, the phenotype of the sasA mutant could suggest that pGpp directly ACTIVATES translation, at least at face-value.

L202 IF2 is not the only translation-relevant target for ppGpp. Protein synthesis is also coupled to nutrient availability through the effect of ppGpp on ribosomal RNA and tRNA synthesis, and on ribosomal assembly factors.

Reviewer #2: In their interesting new Ms, Diez et al follow up their observation of a bimodal distribution of protein synthesis attenuation in a B. subtilis cell population, mediated e.g. by inhibiting IF2, under conditions when the the (p)ppGpp alarmone synthesis is induced (Diez et al. (2020) PNAS 117:15565). B. subtilis cells encode a bifunctional Rel synthetase, which can synthesize pGpp, ppGpp and pppGpp from GMP, GDP or GTP and ATP. Rel contains also a hydrolase domain, allowing the degradation of these alarmones.

In addition, there are also two small alarmone synthetases RelP (YwaC, SasA) & RelQ (YjbM, SasB) present in B. subtilis. The overlapping and also possibly distinct activities of the three different alarmones synthesized by the three different synthetases were recently investigated by different Labs and specifically for cellular pGpp in B. subtilis it was observed that a hydrolase NahA can generate pGpp from (p)ppGpp (Yang et al. (2020) Nat Comms 11:5388).

Here the authors investigated the possible involvement of the two small synthetases SasA and SasB in influencing the observed heterogeneity of protein synthesis detected by OPP labelling (Diez et al. (2020) PNAS 117:15565) (Fig 1A). Interestingly, when sasA was deleted, the translation was inhibited (Fig 1C) in most cells. However, a much higher portion of translating cells were observed in the population when sasB was deleted (Fig 1B).

The experiments in Fig 2 somehow imply that the cells with higher sasA transcription correlate more with the observation of higher translation. The authors argue that therefore differences in SasA expression might be more important for the observed changes in heterogeneity in that growthphase.

Interestingly a synthetase mutant (relAY308A) of Rel, which is probably in general the major source of (p)ppGpp displayed less bimodal distribution with mostly translating cells (Fig 3C), similar to ∆sasB cells (Fig 1B). And the same was true for a SasB variant F42A (Fig 3B), which is known not to be allosterically activated by pppGpp anymore. This allosteric activation results in higher pppGpp synthesis (Steinchen et al (2015) PNAS 112:13348). Both rel and sasB mutations result in more translating cell, consistent with an expected lower alarmone concentration in these strains.

The authors suggest that an allosteric regulation of SasB activity via SasA could explain these results. Since Tagami et al (2012 MicrobiolOpen 1:115) observe an increase of cellular pGpp, they suggest that the pGpp synthesized by SasA might interfere with SasB synthetase activity.

To test this hypothesis, the purified SasB and SasBF42A and show that raising amounts of pGpp somehow result in lowered synthesis of pppGpp. In the control experiment with SasBF42A this is not visible but the pppGpp synthesis is generally lower (Fig4).

Since both Rel and SasB influence SasA the double mutant ∆sasA∆sasB and ∆sasA rel Y308A should abolish the effect of ∆sasA, which can be confirmed experimentally as observed in Fig 5.

Based on these observations the authors suggest that the synthesis of pGpp by SasA negatively influences the Rel mediated activation of SasB and thereby the level of protein synthesis probably via IF2 in a bimodal manner (Fig6).

Comments

-To really give support to this model (Fig 6), it is probably important to measure the different alarmone levels (pGpp, ppGpp, pppGpp) in some of the presented key B. subtilis strains. Since the authors are able to separate the different populations by Fax, it might even be possible to determine this not only in the whole population but also in the respective sub-populations.

-However, it would already help to utilize in addition to the deletion strains of sasA and sasB synthetase defective SasA and SasB variants (e.g. SasAE154V SasBE139V). With such strains it would be easier to conclude that the observed phenotypes of the ∆sasA and/or ∆sasB strains really depend on the synthetase activity of SasA or SasB. And possible protein-protein interactions of these small synthetases, which might also support the observed genetic interaction, would be less perturbed in these strains.

-It is interesting to note that not only Tagami et al observed the in vivo synthesis of pGpp, when SasA was expressed in trans. Fung et al ((2020) Front Mic 11:2083) observed this too. But in addition, they observed a strong pleiotropic effect on the Bacillus physiology and metabolism, which also include the synthesis of other nucleotide second messengers.

Interestingly, Diez et al ((2020) PNAS 117:15565) show themselves in Fig S3 that a SasA induction in trans abolishes the protein synthesis in all the cells of such a population.

I think one could argue that pGpp synthesized by SasA could also directly or indirectly interfere with many cellular processes, which might also include translation.

Therefore, knowing the cellular concentration of pGpp would be important, since it is not clear at what cellular concentration the pGpp could start to influence SasB synthetase activity or the other observed pleiotropic processes.

-Fig 4 The presented in vitro experiments suggest that the pGpp might only diminish the allosteric activation, maybe by somehow competing with pppGpp? Or maybe pGpp competes with substrates?

A more comprehensive analysis and characterization of the SasB enzyme activity and the in vitro influence of pGpp on SasB, would allow to estimate for example determine affinities of pGpp, which would help to understand which cellular concentrations of pGpp have to be reached in vivo.

There seems to be no unit for the amount of pGpp added to the in vitro assay for the SasB activity. What are the concentrations and are they physiologically relevant?

- Fig S6 Both Yang et al and Fung et al observed that NahA is responsible for the generation of more than 80% of the cellular pGpp, possibly from (p)ppGpp synthesized by SasA. Therefore, it is very likely that in the absence of NahA the cellular pGpp concentration in such a strain is much reduced.

However, in Fig S6 the heterogeneity of protein synthesis of the ∆nahA strain looks like the wildtype strain. This suggests that in the absence of NahA the probably strongly reduced cellular pGpp does not make a difference, and might therefore not being majorly involved in generating the observed wild type like heterogeneity in translation.

other comments

Fig 2 The promoter activity might somehow correlate with the translated amount of SasA or SasB, however in the paper of Tagami et al they did a Western blot and showed that they were able to detect SasA only in low levels compared to SasB in the transition growth state.

l55, Intro RsgA might be a misleading example since in B. Subtilis RsgA does not bind ppGpp as shown in the cited paper (8) (Corrigan et al)

line 63-64 – „B. Subtilis RelA primarily generates pppGpp (...)” (11) This reference from Wendrich et al (1997) might not be the best fit, since at that time the two small synthetases were not known yet, and their additional influence could therefore not be considered in this paper at that time.

**********

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Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: None

Reviewer #2: Yes

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PLoS Genet. 2022 May 20;18(5):e1009957. doi: 10.1371/journal.pgen.1009957.r002

Author response to Decision Letter 0

4 Mar 2022

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PLoS Genet. doi: 10.1371/journal.pgen.1009957.r003

Decision Letter 1

Sean Crosson, Lotte Søgaard-Andersen

27 Mar 2022

Dear Dr Dworkin,

This manuscript has been through a second round of review with your original set of reviewers. While some revisions your have made are appreciated, your reviewers again conclude that your conclusion/model are not supported by your data. Though you claim that Figure 6 is not a model, we agree with Reviewer 1 that it will be viewed as such by readers.

After editorial discussion, we agree that significant potential regulatory complexity is not accounted for in the discussion. Keeping Figure 6 in its present form requires some effort to measure nucleotide in the nahA deletion (even at the population level) under the relevant transition condition. If there's a reason why this is not possible, or a reason why such a measurement would be inconclusive, this would be helpful for the editors and reviewers to hear. Experiments that exclude the possibility that SasA or SasB have regulatory activities that do not require their synthase activities also are important if Figure 6 remains in its current form.

To resubmit, use the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.

Yours sincerely,

Sean Crosson

Associate Editor

PLOS Genetics

Lotte Søgaard-Andersen

Section Editor: Prokaryotic Genetics

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The revised ms by Diez et al was again disappointing, with little effort to provide any of the experimental support requested by either reviewer. My main issue still is the oversimplification of the model (although the authors say that Fig. 6 is not a model, most readers will regard it as such). Even though the different (pp)pGpp synthetases may generate preferential products under specific circumstances, they are nonetheless mixed at best, and in some cases not yet completely determined. Despite a minor text modification, IF2 comes across as the only translation target of (p)ppGpp, when several other components of the translational apparatus, including rRNA and tRNA synthesis, are well known to be (p)ppGpp targets. The paper globally, and the schematic in Fig. 6, need be redone to incorporate other possibilities. For example, when a mixture of products is known to be produced, the favored one could have a larger font in Fig. 6. pGpp could have an asterisk to indicate that the inhibitory effect was shown directly, but ppGp should also be shown. rRNA and tRNA should be mentioned along with IF2 as targets of (pp)pGpp.

The product of SasA is either pGpp or ppGp, but more likely ppGp, according to the authors of the cited paper PMID 2950019. For reasons that are unclear, the authors propose to call this mixture pGpp (which I find a little disingenuous), and then only test pGpp (and not ppGp) in Fig. 4 as a competitive inhibitor of the allosteric activation of SasB by pppGpp. The mixture should be referred to as pGpp/ppGp.

As referee 2 pointed out, both Yang et al and Fung et al observed that NahA is responsible for the generation of more than 80% of the cellular pGpp, possibly from (p)ppGpp synthesized by SasA. Therefore, it is very likely that in the absence of NahA the cellular pGpp concentration in such a strain is much reduced and the fact the nahA deletion strain behaves like the wt is not coherent with the model presented. To explain this discrepancy, the authors make the not very convincing suggestion that this may not be the case in late transition phase, but do not provide any evidence to support this. In the absence of experimental evidence, this for me is a potential symptom of an over-simplified model.

I agree with referee 2 that it is important to measure the different nucleotides present in the different mutants under the experimental conditions tested to provide support for the model, even if it is only technically feasible at the population level.

Minor points:

Fig. S1 should be described in the text. The sentences provided L88-91 reference Fig. S2 and still refer to 95% of entire population in late transition phase, when this is clearly not the case.

It is still not clear to me how the quartiles were designated in Figure 2. Only two seem to be delineated in Figure 2A and B. Labelling the quartiles would help. Is only the late transition peak considered? If so how was the growth phase of the two peaks determined?

Abstract (L29) There is one ‘p’ too many in (pp)ppGpp

Reviewer #2: I attached a PDF file of the ReReview text.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Click here for additional data file.^{(63.5KB, pdf)}

PLoS Genet. 2022 May 20;18(5):e1009957. doi: 10.1371/journal.pgen.1009957.r004

Author response to Decision Letter 1

13 Apr 2022

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Submitted filename: response to reviewers.docx

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PLoS Genet. doi: 10.1371/journal.pgen.1009957.r005

Decision Letter 2

Sean Crosson, Lotte Søgaard-Andersen

24 Apr 2022

Dear Dr Dworkin,

We are pleased to inform you that your manuscript entitled "Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Comments from the reviewers (if applicable):

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PLoS Genet. doi: 10.1371/journal.pgen.1009957.r006

Acceptance letter

Sean Crosson, Lotte Søgaard-Andersen

16 May 2022

PGENETICS-D-21-01528R2

Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation

Dear Dr Dworkin,

We are pleased to inform you that your manuscript entitled "Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

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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. Determination of “OFF” cells using stationary phase cells.

(PDF)

Click here for additional data file.^{(29.9KB, pdf)}

S2 Fig. Late transition phase time point and application of “OFF” cutoff to transition phase cells.

(PDF)

Click here for additional data file.^{(67KB, pdf)}

S3 Fig. pGpp does not inhibit SasB basal activity.

(PDF)

Click here for additional data file.^{(51.2KB, pdf)}

S4 Fig. Effect of nahA on heterogeneity.

(A, B) Representative pictures and population distributions of OPP labeled (A) wildtype (JDB1772), (B) ΔnahA (JDB4095) strains during late transition phase.

(PDF)

Click here for additional data file.^{(236.1KB, pdf)}

S1 Table. Plasmids used in this study.

(PDF)

Click here for additional data file.^{(24.1KB, pdf)}

S2 Table. Strains used in this study.

(PDF)

Click here for additional data file.^{(36.7KB, pdf)}

S3 Table. Oligonucleotides used in this study.

(PDF)

Click here for additional data file.^{(19.4KB, pdf)}

Attachment

Submitted filename: Response to Reviewers-3.1.22.pdf

Click here for additional data file.^{(138.8KB, pdf)}

Attachment

Submitted filename: DiezReRev.pdf

Click here for additional data file.^{(63.5KB, pdf)}

Attachment

Submitted filename: response to reviewers.docx

Click here for additional data file.^{(27.3KB, docx)}

Data Availability Statement

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

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PERMALINK

Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation

Simon Diez

Molly Hydorn

Abigail Whalen

Jonathan Dworkin

Roles

Abstract

Author summary

Introduction

Results

The SasA and SasB (pp)pGpp synthases contribute to heterogeneity

Fig 1. sasB and sasA have opposite effects on bimodality.

sasA but not sasB expression is correlated with levels of protein synthesis

Fig 2. Relationship between sasA or sasB expression and OPP incorporation.

SasB allosteric activation is necessary for heterogeneity

Fig 3. Allosteric activation of SasB is required for bimodality during exit from rapid growth.

SasB allosteric activation is inhibited by pGpp

Fig 4. pGpp inhibits the allosteric activation of SasB by pppGpp.

Fig 5. sasA effect is dependent on sasB and relA.

Discussion

Regulation of protein synthesis during nutrient limitation

Physiological sources of variability in SasB activity

Allosteric activation of (pp)pGpp synthases

(pp)pGpp synthases

Physiological implications of heterogeneity in protein synthesis

Materials and methods

Strains and media

Growth curves

OPP labeling

Protein expression and purification

pGpp synthesis

SasB activity assays and TLC

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Sean Crosson

Lotte Søgaard-Andersen

Roles

Author response to Decision Letter 0

Decision Letter 1

Sean Crosson

Lotte Søgaard-Andersen

Roles

Author response to Decision Letter 1

Decision Letter 2

Sean Crosson

Lotte Søgaard-Andersen

Roles

Acceptance letter

Sean Crosson

Lotte Søgaard-Andersen

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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