Kinetic Characterization of the WalRKSpn(VicRK) Two-Component System ofStreptococcus pneumoniae: Dependence of WalKSpn(VicK) Phosphatase Activity on Its PAS Domain

Gutu, Alina D.; Wayne, Kyle J.; Sham, Lok‐To; Winkler, Malcolm E.

doi:10.1128/jb.01690-09

Cited by 71 publications

(122 citation statements)

References 99 publications

(155 reference statements)

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“…At the initial stage, the autophosphorylation reaction is first order in ATP and the dependence of k obs on the ATP concentration was half maximal, at 40 Ϯ 2 M (K ATP ). The overall apparent pseudo-first-order rate of autophosphorylation, K cat (K phos ) is 0.11 Ϯ 0.002 min Ϫ1 , which is similar to that of other histidine kinases, such as S. aureus WalK (13), Streptococcus pneumoniae WalK (29), Bacillus subtilis NarQ (30), and S. aureus VraS (31).…”

Section: Resultsmentioning

confidence: 62%

Influence of the AgrC-AgrA Complex on the Response Time of Staphylococcus aureus Quorum Sensing

et al. 2014

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The Staphylococcus aureus agr quorum-sensing system plays a major role in the transition from the persistent to the virulent phenotype. S. aureus agr type I to IV strains are characterized by mutations in the sensor domain of the histidine kinase AgrC and differences in the sequences of the secreted autoinducing peptides (AIP). Here we demonstrate that interactions between the cytosolic domain of AgrC (AgrC Cyto ) and the response regulator domain of AgrA (AgrA RR ) dictate the spontaneity of the cellular response to AIP stimuli. The crystal structure of AgrC Cyto provided a basis for a mechanistic model of AgrC-AgrA interactions. This model enabled an analysis of the biochemical and biophysical parameters of AgrC-AgrA interactions in the context of the conformational features of the AgrC-AgrA complex. This analysis revealed distinct sequence and conformational features that determine the affinity, specificity, and kinetics of the phosphotransfer reaction. This step, which governs the response time for transcriptional reengineering triggered by an AIP stimulus, is independent of the agr type and similar for agonist and antagonist stimuli. These experimental data could serve as a basis on which to validate simulations of the quorum-sensing response and for strategies that employ the agr quorum-sensing system to combat biofilm formation in S. aureus infections. Multiple pore-forming toxins, immune evasion factors, and adhesins contribute to acute and chronic Staphylococcus aureus infections in humans (1). The expression of most of these virulence factors is controlled by the accessory gene regulator (agr), a two-component regulatory system in S. aureus (2). The sequence composition of the autoinducing peptide (AIP) with a five-member thiolactone ring provides a basis on which to distinguish between different S. aureus strains, also referred to as agr types I to IV. The interaction of the AIP with AgrC, a membranebound histidine kinase, regulates the catalytic activity of AgrC. Mutations in AgrC across S. aureus strains of different agr types are confined to the ectodomain. This observation is consistent with experimental data that suggest that the signal recognition mechanism is confined to the extracellular and membrane-associated components (3). The subsequent steps of signal transduction involve phosphorylation of AgrA by AgrC, followed by the interaction of phosphorylated AgrA with cognate promoters to trigger agr-dependent reengineering of the transcriptional profile. This step incorporates signal amplification due to AgrA-induced expression from the P2 promoter and expression of RNAIII, a downstream effector that regulates the expression of virulence factors and other transcriptional regulators (4). Prominent changes in the transcriptional profile induced by AgrA include the expression of the characterized immune response suppressors ␣ and ␤ phenol-soluble modulins (5). The production and secretion of the AIPs involve the action of the permease AgrB on the AgrD propeptide (6).The sensor module of AgrC (resid...

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Section: Resultsmentioning

confidence: 62%

Influence of the AgrC-AgrA Complex on the Response Time of Staphylococcus aureus Quorum Sensing

et al. 2014

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“…These studies revealed that VicK is a bifunctional sensor protein that alternates kinase and phosphatase activities, and that the phosphatase activity is dependent on the PAS domain [102,136]. There is also evidence that both forms of VicR, phosphorylated (P-VicR) and non-phosphorylated (NP-VicR), are capable of directly binding to a different set of target genes.…”

Section: What Are the Signals Sensed By Vicrk Tcs?mentioning

confidence: 99%

Two-component signal transduction systems in oral bacteria

Mattos-Graner

Duncan²

2017

Journal of Oral Microbiology

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We present an overview of how members of the oral microbiota respond to their environment by regulating gene expression through two-component signal transduction systems (TCSs) to support conditions compatible with homeostasis in oral biofilms or drive the equilibrium toward dysbiosis in response to environmental changes. Using studies on the sub-gingival Gram-negative anaerobe Porphyromonas gingivalis and Gram-positive streptococci as examples, we focus on the molecular mechanisms involved in activation of TCS and species specificities of TCS regulons.

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“…Since WalK Spn has both autokinase and phosphorylated WalR Spn (WalR Spn ϳP) phosphatase activities (19), this excess implies that WalRK Spn may be a robust signaling system (3,18). The impact of these cellular amounts and ratios to signal transduction by the WalRK Spn TCS awaits determinations of binding constants, kinetic properties, and cellular phosphorylation levels.…”

Section: Fig 2 Localization Of Walk Spn -(C)-flag Walr Spn -(C)-l-mentioning

confidence: 99%

“…4A). As a standard, we spiked various concentrations of purified (N)-His 10 -WalR Spn protein (19,31) (see Table S2 in the supplemental material) into an extract of a ⌬walR Spn P c -pcsB ϩ mutant (IU1473 , Table 1) containing the same amount of total protein as that used for the walR Spn ϩ strain IU4062 (Table 1). Unencapsulated mutants that are predominantly diplococci instead of chains of cells were used in these experiments to simplify estimates of cellular amounts (2).…”

Section: Fig 2 Localization Of Walk Spn -(C)-flag Walr Spn -(C)-l-mentioning

confidence: 99%

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Localization and Cellular Amounts of the WalRKJ (VicRKX) Two-Component Regulatory System Proteins in Serotype 2Streptococcus pneumoniae

et al. 2010

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The WalRK two-component regulatory system coordinates gene expression that maintains cell wall homeostasis and responds to antibiotic stress in low-GC Gram-positive bacteria. Phosphorylated WalR (VicR) of the major human respiratory pathogen Streptococcus pneumoniae (WalR Spn ) positively regulates transcription of several surface virulence genes and, most critically, pcsB, which encodes an essential cell division protein. Despite numerous studies of several species, little is known about the signals sensed by the WalK histidine kinase or the function of the WalJ ancillary protein encoded in the walRK Spn operon. To better understand the functions of the WalRKJ Spn proteins in S. pneumoniae, we performed experiments to determine their cellular localization and amounts. In contrast to WalK from Bacillus subtilis (WalK Bsu ), which is localized at division septa, immunofluorescence microscopy showed that WalK Spn is distributed throughout the cell periphery. WalJ Spn is also localized to the cell surface periphery, whereas WalR Spn was found to be localized in the cytoplasm around the nucleoid. In fractionation experiments, WalR Spn was recovered from the cytoplasmic fraction, while WalK Spn and the majority of WalJ Spn were recovered from the cell membrane fraction. This fractionation is consistent with the localization patterns observed. Lastly, we determined the cellular amounts of WalRKJ Spn by quantitative Western blotting. The WalR Spn response regulator is relatively abundant and present at levels of Ϸ6,200 monomers per cell, which are Ϸ14-fold greater than the amount of the WalK Spn histidine kinase, which is present at Ϸ460 dimers (920 monomers) per cell. We detected Ϸ1,200 monomers per cell of WalJ Spn ancillary protein, similar to the amount of WalK Spn .

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Kinetic Characterization of the WalRK_Spn(VicRK) Two-Component System ofStreptococcus pneumoniae: Dependence of WalK_Spn(VicK) Phosphatase Activity on Its PAS Domain

Cited by 71 publications

References 99 publications

Influence of the AgrC-AgrA Complex on the Response Time of Staphylococcus aureus Quorum Sensing

Influence of the AgrC-AgrA Complex on the Response Time of Staphylococcus aureus Quorum Sensing

Two-component signal transduction systems in oral bacteria

Localization and Cellular Amounts of the WalRKJ (VicRKX) Two-Component Regulatory System Proteins in Serotype 2Streptococcus pneumoniae

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