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Crosstalk between guanosine nucleotides regulates cellular heterogeneity in protein synthesis during nutrient limitation [1]

['Simon Diez', 'Department Of Microbiology', 'Immunology', 'College Of Physicians', 'Surgeons', 'Columbia University', 'New York', 'United States Of America', 'Molly Hydorn', 'Abigail Whalen']

Date: 2022-07

(A, B, C) Representative pictures and population distributions of OPP labeled (A) wildtype (JDB1772), (B) ΔsasB (JDB4310) and (C) ΔsasA (JDB4311) during late transition phase. % of population “OFF” as determined in S1 Fig presented above each representative distribution. Statistical significance was determined by comparing three independent populations of WT to either mutant. P-values are 0.046 and 0.011 for ΔsasB and ΔsasA respectively.

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 ])

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 (OD 600 ~0.685) as “OFF” ( S2 Fig ). By convention, we define the remainder of the population as “ON.”

(A, B) Representative population distribution of B. subtilis carrying a transcriptional reporter of (A) P sasB -yfp (JDB4341) or (B) P sasA -yfp (JDB4030) during exponential (light blue/green) and late transition phase (dark blue/green). Black lines represent quartiles used in C and D. (C, D) Average OPP incorporation in late transition phase of each quartile of (C) P sasB -yfp expression or (D) P sasA -yfp expression from lowest to highest. Statistical analysis (one tailed t-test) showed no significant difference in OPP incorporation between any P sasB -yfp quartiles (p>0.05) and significantly higher OPP incorporation between quartiles 1 and 3 and quartiles 1 and 4 of P sasA -yfp expression (p-values 0.027 and 0.016, respectively).

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.

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.

(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.

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.

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 SasBF42 mutant protein that is insensitive to allosteric activation by pppGpp [16]. As reported SasBF42A 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 SasBF42A even when pppGpp is included (Fig 4B).

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TIFF original image Download: Fig 4. pGpp inhibits the allosteric activation of SasB by pppGpp. (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 SasBF42A 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. https://doi.org/10.1371/journal.pgen.1009957.g004

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 RelAY308A and carrying a ΔsasA mutation exhibited a loss of heterogeneity in protein synthesis similar to the relAY308A 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.

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1009957

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