Instability of Acetate Kinase Mutants and Their Effects on Acetyl Phosphate and ATP Amounts in Streptococcus pneumoniae
Ontology highlight
ABSTRACT:
ORGANISM(S): Streptococcus pneumoniae
PROVIDER: GSE23404 | GEO | 2010/11/29
SECONDARY ACCESSION(S): PRJNA131053
REPOSITORIES: GEO
Similar Datasets
Project description:Pyruvate oxidase encoded by spxB is a major virulence factor in the human respiratory pathogen Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes large amounts of H2O2 and acetyl phosphate, which can serve as a phosphoryl group donor to response regulators and be converted to ATP. SpxB is the main source of the millimolar concentrations of H2O2 produced and tolerated by pneumococcus, despite its lack of a catalase. We report here the first cis- and trans-acting regulatory elements for spxB transcription. These elements were identified in a genetic screen, similar to those used previously for phase variants, for spontaneous mutations that caused colonies of virulent serotype 2 strain D39 to change from a transparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency of 3 x 10-5) were impaired for SpxB function. Modeling suggested that two mutations changed amino acids in SpxB required for FAD cofactor or subunit binding. One mutation deleted a cis-acting adjacent direct repeat required for optimal spxB transcription. The other three independent mutations created the same frameshift near the start of a trans-acting regulatory gene designated as spxR. The SpxR protein contains helix-turn-helix, CBS, and HotDog domains implicated in DNA, adenosine, and CoA compound binding, respectively, consistent with the idea that SpxR positively regulates spxB transcript amount in response to energy and metabolic state rather than oxidative state. Finally, microarray analyses of a null spxB or a spxR mutant revealed the presence of a new oxidative stress response in pneumococcus and unexpectedly demonstrated that SpxR strongly positively regulates the transcript amount of the strH exoglycosidase gene, which like spxB, has been implicated in host colonization. Keywords: genetic modification Bacterial strains were grown exponentially in rich (BHI) media at 37C and an atmosphere of 5% CO2, and were processed as described in the related Sample records. Samples were collected from three independent biological replicates and included one dye swap. Data were normalized using the Lowess (subgrid) method without background subtraction.
2010-05-26 | E-GEOD-7109 | biostudies-arrayexpress
Project description:Pyruvate oxidase encoded by spxB is a major virulence factor in the human respiratory pathogen Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes large amounts of H2O2 and acetyl phosphate, which can serve as a phosphoryl group donor to response regulators and be converted to ATP. SpxB is the main source of the millimolar concentrations of H2O2 produced and tolerated by pneumococcus, despite its lack of a catalase. We report here the first cis- and trans-acting regulatory elements for spxB transcription. These elements were identified in a genetic screen, similar to those used previously for phase variants, for spontaneous mutations that caused colonies of virulent serotype 2 strain D39 to change from a transparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency of 3 x 10-5) were impaired for SpxB function. Modeling suggested that two mutations changed amino acids in SpxB required for FAD cofactor or subunit binding. One mutation deleted a cis-acting adjacent direct repeat required for optimal spxB transcription. The other three independent mutations created the same frameshift near the start of a trans-acting regulatory gene designated as spxR. The SpxR protein contains helix-turn-helix, CBS, and HotDog domains implicated in DNA, adenosine, and CoA compound binding, respectively, consistent with the idea that SpxR positively regulates spxB transcript amount in response to energy and metabolic state rather than oxidative state. Finally, microarray analyses of a null spxB or a spxR mutant revealed the presence of a new oxidative stress response in pneumococcus and unexpectedly demonstrated that SpxR strongly positively regulates the transcript amount of the strH exoglycosidase gene, which like spxB, has been implicated in host colonization. Keywords: genetic modification
2008-02-22 | GSE7109 | GEO
Project description:The catalase-negative, facultative anaerobe Streptococcus pneumoniae D39 is naturally resistant to hydrogen peroxide (H2O2) produced endogenously by pyruvate oxidase (SpxB). Here, we investigate the adaptive response to endogenously produced H2O2. We show that lactate oxidase, which converts lactate to pyruvate, positively impacts pyruvate flux through SpxB and that ∆lctO mutants produce significantly lower H2O2. In addition, both the SpxB and pyruvate dehydrogenase complex (PDHC) pathways contribute to acetyl-CoA production during aerobic growth, and the pyruvate format lyase (PFL) pathway is the major acetyl-CoA pathway during anaerobic growth. Microarray analysis of the D39 strain cultured under aerobic vs. strict anaerobic conditions show up-regulation of spxB, a rhodanese-like protein (spd0091), tpxD, sodA, piuB, piuD and an Fe-S protein biogenesis operon under H2O2-producing conditions. Proteome profiling of H2O2-induced sulfenylation reveals that sulfenylation levels correlate with cellular H2O2 production, with endogenous sulfenylation of ≈50 proteins. Deletion of tpxD increases cellular sulfenylation 5-fold and has an inhibitory effect on ATP generation. Two major targets of protein sulfenylation are glyceraldehyde-3-phosphate dehydrogenase (GapA) and SpxB itself, but also include pyruvate kinase, LctO, AdhE and acetate kinase (AckA). Sulfenylation of GapA is inhibitory, while the effect on SpxB activity is negligible, consistent with this cell-abundant protein functioning as a “sink” for endogenous H2O2. Strikingly, four enzymes of capsular polysaccharide biosynthesis are sulfenylated, as are enzymes associated nucleotide biosynthesis via ribulose-5-phosphate. We propose that LctO/SpxB-generated H2O2 functions as a signaling molecule to down-regulate capsule production and drive altered flux through sugar utilization pathways.
2016-12-08 | GSE19791 | GEO
Project description:We applied SD-SILAC to determine absolute acetylation stoichiometry in exponentially-growing and stationary-phase wild type and sirtuin deacetylase CobB-deficient E. coli. We further assayed stoichiometry in phophotransacetylase (pta) and acetate kinase (ackA) mutant strains in the presence and absence of the sirtuin inhibitor nicotinamide. Comparison to relative quantification under the same conditions validated our stoichiometry estimates at hundreds of sites, demonstrating the accuracy of our method. We measured acetylation stoichiometry at 3,669 unique sites and found that the vast majority of acetylation occurs at very low stoichiometry (median 0.04%). Manipulations that cause increased nonenzymatic acetylation by acetyl-phosphate (AcP), such stationary-phase arrest and deletion of ackA, resulted in globally increased acetylation stoichiometry. Similar to sirtuin deacetylase 3 (SIRT3) in mitochondria, CobB suppressed acetylation to lower than median stoichiometry at hundreds of sites in WT, pta, and ackA cells. These results suggest an evolutionarily conserved function of sirtuin deacetylases in suppressing low stoichiometry acetylation.
2017-03-07 | PXD005757 | Pride
Project description:spxB-encoded pyruvate oxidase is a major virulence factor of Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes H2O2 and acetyl phosphate, which play roles in metabolism, signaling, and oxidative stress. We report here the first cis- and trans-acting regulatory elements for spxB transcription. These elements were identified in a genetic screen for spontaneous mutations that caused colonies of strain D39 to change from a semi-transparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency 3x10-5) were impaired for SpxB function or expression. Two mutations changed amino acids in SpxB likely required for cofactor or subunit binding. One mutation defined a cis-acting adjacent direct repeat required for optimal spxB transcription. The other three spontaneous mutations created the same frameshift near the start of the trans-acting spxR regulatory gene. The SpxR protein contains helix-turn-helix, CBS, and HotDog domains implicated in binding DNA, adenosyl compounds, and CoA-containing compounds, respectively, and suggest that SpxR positively regulates spxB transcription in response to energy and metabolic state. Finally, microarray analyses of a spxR mutant demonstrated that SpxR positively regulates the strH exoglycosidase gene, which like spxB, has been implicated in colonization. Keywords: bacterial genetic modification
2008-01-04 | GSE9284 | GEO
Project description:Transcriptional comparisons between wild-type Streptococcus mutans UA159 and two mutant strains (∆ackA and ∆ackA pta).
2013-05-11 | GSE46835 | GEO
Project description:We compared the gene expression patterns of macrophages infected with S. oralis wild type and SpxB KO, a strain that does not produce H2O2.
2017-12-01 | GSE94317 | GEO
Project description:A Synthetic Gene-Metabolic Oscillator
Reference:
Fung et al; Nature (2005) 435:118-122
Name of kinetic law
Reaction
Glycolytic flux, V_gly:
nil -> AcCoA;
Flux to TCA cycle/ETOH, V_TCA:
AcCoA -> TCA/EtOH;
HOAc ex/import,reversible, V_out:
HOAc -> HOAc_E
V_Pta:
AcCoA + Pi -> AcP + CoA
reversible, V_Ack:
AcP + ADP -> OAc + ATP
V_Acs:
OAC + ATP -> AcCoA +PPi
Acetic acid-base equillibrium, reversible, V_Ace:
OAC + H -> HOAc
Expression of LacI, R_LacI:
nil -> LacI
Expression of Acs, R_Acs:
nil -> Acs
Expression of Pta, R_Pta:
nil -> Pta
LacI degradation, R_dLacI:
LacI -> nil
Acs degradation, R_dAcs:
Acs -> nil
Pta degradation, R_dPta:
Pta -> nil
For this model the differential equation for V_Ace was changed from:
C*(AcP*H-K_eq*OAC) with C = 100 in the supplemental material
to C*(OAc*H-K_eq*HOAc) with C = 100, as in
Bulter et. al; PNAS(2004),101,2299-2304
, and a value for K_eq of 5*10^-4 after communication with the authors.
translated to SBML by:
Lukas Endler(luen at tbi.univie.ac.at), Christoph Flamm (xtof at tbi.univie.ac.at)
Biomodels Curation
The model reproduces 3a of the paper for glycolytic flux Vgly = 0.5. The authors have agreed that the values on Y-axis are marked wrong and hence there is a discrepancy between model simulation results and the figure. Also, note that the values of concentration and time are in dimensionless units. The model was successfully tested on MathSBML and Jarnac.
2024-09-02 | BIOMD0000000067 | BioModels
Project description:We studied the transcriptomic response of S. pneumoniae to moxifloxacin, a fluoroquinolone that inhibits DNA gyrase. At 10 × MIC, 30 and 140 responsive genes were detected at 15 and 30 minutes, respectively. Several pathways leading to an increase in pyruvate showed up-regulation. These included 2 genes introducing P-sugars into the glycolysis and 3 out of 7 genes of the glycolysis pathway, which converts fructose-6P to pyruvate. In addition, an increase in acetyl-coA would be expected from the down-regulation of the genes coding for the acetyl-coA carboxylase, and in turn, to a further increase in pyruvate by the up-regulation of formate acetyltransferase. Since pyruvate is converted to hydrogen peroxide (H2O2) by pyruvate oxidase (SpxB), its increase would lead to an equivalent increase in the intracellular amount of H2O2, and in turn, in those of hydroxyl radicals resulting from the Fenton reaction, which damage DNA, lipids and proteins. We observed similar increases in the production of H2O2 and hydroxyl radicals under moxifloxacin treatment. These reactive oxygen species contributed to the lethality of the drug, as showed by the attenuation of its lethality in a strain lacking SpxB. These results support the production of redox alterations by fluoroquinolones and are in agreement with our previous findings showing that levofloxacin, an inhibitor of topoisomerase IV, triggers the transcriptional activation of iron transport genes. Both fluoroquinolones stimulate the Fenton reaction in their mechanism of action, by increasing the amounts of any of their two components: iron by levofloxacin or H2O2 by moxifloxacin.
2016-02-05 | GSE68947 | GEO
Project description:Our understanding of the synergism between S. pneumoniae and influenza virus remains incomplete. The classic dogma has been that influenza attenuates the host innate immunity and increase the susceptibility to subsequent bacterial infection. Therefore, the majority of current studies have been focusing on the interaction of S. pneumoniae and influenza in the context of host cells. By contrast, in this study, we set out to investigate the response of pneumococcus alone to virus infection. Our hypothesis was that prior to causing any damages to host cells, influenza may have induced (lethal) changes to pneumococcus cell itself. Indeed, a very recent evidence has shown that direct viral treatment to pneumococcus will increase its adhesion to macrophage cells. Here, using quantitative shotgun approach, we attempt to investigate the proteomic alterations of S. pneumoniae by influenza virus challenge, and provide a landscape of interactions between the IAV and pneumococcus.
2022-12-09 | PXD016122 | Pride