Disruption of the alsSD operon of Enterococcus faecalis impairs growth on pyruvate at low pH

Repizo, Guillermo Daniel; Mortera, Pablo; Magni, Christian

doi:10.1099/mic.0.047662-0

Cited by 22 publications

(15 citation statements)

References 40 publications

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“…This effectively minimizes the carbon diverted to the generation of toxic acetate through the CidC pathway. More importantly evidence presented here also shows that the AlsSD pathway consumes protons from the cytoplasm and helps maintain pH homeostasis similar to the enterococci and lactobacilli [21], [30]. We contend that the ability of AlsSD to antagonize CidC activity effectively results in the modulation of intracellular pH and constitutes a robust mechanism to limit cell death and optimize biomass within the microenvironment of a microcolony.…”

Section: Discussionmentioning

confidence: 64%

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A Central Role for Carbon-Overflow Pathways in the Modulation of Bacterial Cell Death

et al. 2014

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Similar to developmental programs in eukaryotes, the death of a subpopulation of cells is thought to benefit bacterial biofilm development. However mechanisms that mediate a tight control over cell death are not clearly understood at the population level. Here we reveal that CidR dependent pyruvate oxidase (CidC) and α-acetolactate synthase/decarboxylase (AlsSD) overflow metabolic pathways, which are active during staphylococcal biofilm development, modulate cell death to achieve optimal biofilm biomass. Whereas acetate derived from CidC activity potentiates cell death in cells by a mechanism dependent on intracellular acidification and respiratory inhibition, AlsSD activity effectively counters CidC action by diverting carbon flux towards neutral rather than acidic byproducts and consuming intracellular protons in the process. Furthermore, the physiological features that accompany metabolic activation of cell death bears remarkable similarities to hallmarks of eukaryotic programmed cell death, including the generation of reactive oxygen species and DNA damage. Finally, we demonstrate that the metabolic modulation of cell death not only affects biofilm development but also biofilm-dependent disease outcomes. Given the ubiquity of such carbon overflow pathways in diverse bacterial species, we propose that the metabolic control of cell death may be a fundamental feature of prokaryotic development.

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Section: Discussionmentioning

confidence: 64%

“…Intracellular pH was determined as previously described [30] with the minor modifications. Briefly, bacterial cells were grown in TSB (35 mM glucose) and 1 mL was harvested by centrifugation upon reaching an OD 600 of 2.…”

Section: Methodsmentioning

confidence: 99%

A Central Role for Carbon-Overflow Pathways in the Modulation of Bacterial Cell Death

et al. 2014

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“…The cellular toxicity attributed to pyruvate under acid stress is controversial. While some studies point to a beneficial role for intracellular pyruvate following acidic stress, others have alluded to a more toxic nature for this metabolite (Harvey & Collins, 1963;Tsau et al, 1992;Repizo et al, 2011;Wu et al, 2014). Hence, to test whether intracellular pyruvate affects S. aureus under acidic stress, we challenged the wild-type strain with increasing concentrations of sodium pyruvate in Tryptic Soy broth (TSB) that was acidified to an initial pH of 4.5 and monitored growth at a wavelength of 600 nm (OD 600 ) for 24 h. Because of the weak acid properties of pyruvic acid (pKa5 2.49), this approach not only allows a small percentage (<1%; estimate based on the Henderson-Hasselbalch equation) of extracellular pyruvic acid to diffuse freely into cells and ensure cytoplasmic acidification, but also simultaneously increases the levels of intracellular pyruvate (upon disassociation of pyruvic acid within the cytoplasm) in the acid stressed cells.…”

Section: Resultsmentioning

confidence: 99%

The LysR‐type transcriptional regulator, CidR, regulates stationary phase cell death in Staphylococcus aureus

Chaudhari

Thomas

Sadykov

et al. 2016

Molecular Microbiology

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Summary The Staphylococcus aureus LysR-type transcriptional regulator, CidR, activates the expression of two operons including cidABC and alsSD that display pro- and anti-death functions, respectively. Although several investigations have focused on the functions of different genes associated with these operons, the collective role of the CidR regulon in staphylococcal physiology is not clearly understood. Here we reveal that the primary role of this regulon is to limit acetate-dependent potentiation of cell death in staphylococcal populations. Although both CidB and CidC promote acetate generation and cell death, the CidR-dependent co-activation of CidA and AlsSD counters the effects of CidBC by redirecting intracellular carbon flux towards acetoin formation. From a mechanistic standpoint, we demonstrate that CidB is necessary for full activation of CidC, whereas CidA limits the abundance of CidC in the cell.

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“…Assays were conducted according to the manufacturer’s protocol using a 96-well plate format. Acetoin production was measured using a Voges-Proskauer test adapted for use in a 96-well plate as described previously (66). For all analyses, cells were grown to early log, mid-log, and late log phase in FMCG as described above.…”

Section: Methodsmentioning

confidence: 99%

Basal Levels of (p)ppGpp in Enterococcus faecalis: the Magic beyond the Stringent Response

Gaca

Kajfasz

Miller

et al. 2013

mBio

112

180

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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)ppGpp0] 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)ppGpp0 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.

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Disruption of the alsSD operon of Enterococcus faecalis impairs growth on pyruvate at low pH

Cited by 22 publications

References 40 publications

A Central Role for Carbon-Overflow Pathways in the Modulation of Bacterial Cell Death

A Central Role for Carbon-Overflow Pathways in the Modulation of Bacterial Cell Death

The LysR‐type transcriptional regulator, CidR, regulates stationary phase cell death in Staphylococcus aureus

Basal Levels of (p)ppGpp in Enterococcus faecalis: the Magic beyond the Stringent Response

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