Basal levels of (p)ppGpp in Enterococcus faecalis: the magic beyond the stringent response

Abstract

The stringent response (SR), mediated by the alarmone (p)ppGpp, is a conserved bacterial adaptation system controlling broad metabolic alterations necessary for survival under adverse conditions. In Enterococcus faecalis, production of (p)ppGpp is controlled by the bifunctional protein RSH (for "Rel SpoT homologue"; also known as RelA) and by the monofunctional synthetase RelQ. Previous characterization of E. faecalis strains lacking rsh, relQ, or both revealed that RSH is responsible for activation of the SR and that alterations in (p)ppGpp production negatively impact bacterial stress survival and virulence. Despite its well-characterized role as the effector of the SR, the significance of (p)ppGpp during balanced growth remains poorly understood. Microarrays of E. faecalis strains producing different basal amounts of (p)ppGpp identified several genes and pathways regulated by modest changes in (p)ppGpp. Notably, expression of numerous genes involved in energy generation were induced in the rsh relQ [(p)ppGpp(0)] strain, suggesting that a lack of basal (p)ppGpp places the cell in a "transcriptionally relaxed" state. Alterations in the fermentation profile and increased production of H2O2 in the (p)ppGpp(0) strain substantiate the observed transcriptional changes. We confirm that, similar to what is seen in Bacillus subtilis, (p)ppGpp directly inhibits the activity of enzymes involved in GTP biosynthesis, and complete loss of (p)ppGpp leads to dysregulation of GTP homeostasis. Finally, we show that the association of (p)ppGpp with antibiotic survival does not relate to the SR but rather relates to basal (p)ppGpp pools. Collectively, this study highlights the critical but still underappreciated role of basal (p)ppGpp pools under balanced growth conditions.

Importance: Drug-resistant bacterial infections continue to pose a significant public health threat by limiting therapeutic options available to care providers. The stringent response (SR), mediated by the accumulation of two modified guanine nucleotides collectively known as (p)ppGpp, is a highly conserved stress response that broadly remodels bacterial physiology to a survival state. Given the strong correlation of the SR with the ability of bacteria to survive antibiotic treatment and the direct association of (p)ppGpp production with bacterial infectivity, understanding how bacteria produce and utilize (p)ppGpp may reveal potential targets for the development of new antimicrobial therapies. Using the multidrug-resistant pathogen Enterococcus faecalis as a model, we show that small alterations to (p)ppGpp levels, well below concentrations needed to trigger the SR, severely affected bacterial metabolism and antibiotic survival. Our findings highlight the often-underappreciated contribution of basal (p)ppGpp levels to metabolic balance and stress tolerance in bacteria.

FIG 1

Determination of basal (p)ppGpp levels during nonstressed growth. (A) 2D TLC of [³²P]orthophosphate labeled cells. Cells were grown in low-phosphate FMCG to an OD₆₀₀ of 0.25, labeled with 150 µCi ml⁻¹ of ³²P, and grown to a final optical density (600 nm) of 0.4. The identity of the spot appearing at the top left quadrant of each TLC plate is unknown. (B) Fold change of guanine nucleotide pools compared to the wild-type from 1D TLC separation of ³²P-labeled cell extracts. GTP, ppGpp, and pppGpp spots were quantified using a phosphorimager.

FIG 2

Genes and pathways induced in the (p)ppGpp⁰ strain that lead to the production of pyruvate and heterolactic fermentation products derived from pyruvate in E. faecalis. Shaded pathways or proteins specify activated genes detected from microarray analysis. Proteins and protein complexes responsible for the chemical conversion of pyruvate into the five different fermentation end products are in bold but were not detected in microarray comparisons. Dashed lines represent multistep processes. Oad, oxaloacetate dehydrogenase complex; Cit, citrate lyase complex; CitH, citrate transporter; CitM, malate dehydrogenase; Sdh, l-serine dehydratase complex; GlpF, glycerol permease; GlpO, glycerol phosphate oxidase.

FIG 3

Metabolic profile of wild-type OG1RF and (p)ppGpp⁰ strains. The soluble fraction of whole-cell lysates (for pyruvate measurements) or culture supernatants (for fermentation end products) from E. faecalis grown in FMCG were harvested by centrifugation at the indicated optical densities (600 nm) and used to determine the concentrations of pyruvate (A), lactate (B), formate (C), ethanol (D), and acetoin (E) and the culture pH (F). For all assays, n = 6 (*, P < 0.032; **, P < 0.005; ***, P < 0.0001).

FIG 4

Enhanced H₂O₂ production by the (p)ppGpp⁰ strain. Cells of E. faecalis OG1RF and (p)ppGpp⁰ strains grown in FMCG were harvested at the indicated optical densities and washed in PBS buffer. The washed cell suspension was mixed with an equal volume of buffer to determine H₂O₂ production over a 30-min incubation period. For all assays, n = 9 (*, P < 0.0001).

FIG 5

Dysregulation of guanosine metabolism in the (p)ppGpp⁰ strain. (A) (Top) HprT inhibition by increasing concentrations of pppGpp in vitro. Error bars represent standard errors of three independent experiments. (Bottom) Relative enzymatic activity of HprT and Gmk in the presence of GTP or pppGpp. (B) TLC of ³²P-labeled cells showing GTP accumulation in the presence of exogenous guanosine. Cells were grown in FMCG lacking nucleobase supplementation to an OD₆₀₀ of 0.3, labeled with 150 µCi ml⁻¹ of ³²P, and treated with 2 mM guanosine (rG) for 15 min. Control (Ctrl) samples were not treated with guanosine. (C) Growth inhibition of the (p)ppGpp⁰ strain caused by addition of excess guanosine. Cells were grown to early log phase in complete FMCG and diluted into FMCG lacking exogenous nucleobases supplemented with 1 mM guanosine (n = 3).

FIG 6

Antibiotic survival of OG1RF, Δrsh, ΔrelQ, and (p)ppGpp0 strains. Exponentially grown cultures were diluted in fresh FMCG to 5 × 10⁶ to 1 × 10⁷ CFU ml⁻¹, and cell survival after addition of ampicillin (8 µg ml⁻¹) (A), norfloxacin (64 µg ml⁻¹) (B), or chloramphenicol (16 µg ml⁻¹) (C) was monitored over time.