scholarly journals Autoregulation of theS. mutansSloR metalloregulator is constitutive and driven by an independent promoter

2018 ◽  
Author(s):  
Patrick Monette ◽  
Richard Brach ◽  
Annie Cowan ◽  
Roger Winters ◽  
Jazz Weisman ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Metal Ion ◽  
Bacterial Species ◽  
Ion Homeostasis ◽  
Tooth Decay ◽  
Mobility Shift ◽  
Metal Ion Homeostasis ◽  
Metalloregulatory Protein ◽  
Dental Cavities

AbstractStreptococcus mutans, one of ∼600 bacterial species in the human oral cavity, is among the most acidogenic constituents of the plaque biofilm. Considered to be the primary causative agent of dental caries,S. mutansharbors a 25kDa SloR metalloregulatory protein which controls metal ion transport across the bacterial cell membrane to maintain essential metal ion homeostasis. The expression of SloR derives, in part, from transcriptional readthrough of thesloABCoperon which encodes a Mn2+/Fe2+ABC transport system. Herein, we describe the details of thesloABCpromoter that drives this transcription, as well as a novel independent promoter in an intergenic region (IGR) that contributes to downstreamsloRexpression. RT-PCR studies supportsloRtranscription that is independent ofsloABCexpression, and the results of 5′ RACE revealed asloRtranscription start site in the IGR from which the −10 and −35 promoter regions were predicted. The results of gel mobility shift assays support direct SloR binding to the IGR, albeit with lower affinity than SloR binding to thesloABCRpromoter. Function of thesloRpromoter was validated in qRT-PCR experiments. Interestingly,sloRexpression was not significantly impacted when grown in the presence of high manganese, whereas expression of thesloABCoperon was repressed under these conditions. The results ofin vitrotranscription studies support SloR-mediated transcriptional-activation ofsloRand -repression ofsloABC.Taken together, these findings implicate SloR as a bifunctional regulator that repressessloABCpromoter activity and encouragessloRtranscription from an independent promoter.ImportanceTooth decay is a ubiquitous infectious disease that is especially pervasive in underserved communities worldwide.S. mutans-induced carious lesions cause functional, physical, and/or aesthetic impairment in the vast majority of adults, and in 60-90% of schoolchildren in industrialized countries. Billions of dollars are spent annually on caries treatment, and productivity losses due to absenteeism from the workplace are significant. Research aimed at alleviatingS. mutans-induced tooth decay is important because it can address the socioeconomic disparity that is associated with dental cavities and improve overall general health which is inextricably linked to oral health. Research focused on theS. mutansSloR metalloregulatory protein can guide the development of novel therapeutics and so alleviate the burden of dental cavities.

scholarly journals Autoregulation of theStreptococcus mutansSloR Metalloregulator Is Constitutive and Driven by an Independent Promoter

2018 ◽  
Vol 200 (14) ◽  
Author(s):  
Patrick Monette ◽  
Richard Brach ◽  
Annie Cowan ◽  
Roger Winters ◽  
Jazz Weisman ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Metal Ion ◽  
Bacterial Species ◽  
Ion Homeostasis ◽  
Manganese Concentration ◽  
Tooth Decay ◽  
Content Type ◽  
Metal Ion Homeostasis ◽  
Metalloregulatory Protein ◽  
Dental Cavities

ABSTRACTStreptococcus mutans, one of ∼600 bacterial species in the human oral cavity, is among the most acidogenic constituents of the plaque biofilm. Considered to be the primary causative agent of dental caries,S. mutansharbors a 25-kDa SloR metalloregulatory protein which controls metal ion transport across the bacterial cell membrane to maintain essential metal ion homeostasis. The expression of SloR derives in part from transcriptional readthrough of thesloABCoperon, which encodes a Mn2+/Fe2+ABC transport system. Here we describe the details of thesloABCpromoter that drives this transcription as well as those for a novel independent promoter in an intergenic region (IGR) that contributes to downstreamsloRexpression. Reverse transcriptase PCR (RT-PCR) studies support the occurrence ofsloRtranscription that is independent ofsloABCexpression, and the results of 5′ rapid amplification of cDNA ends (5′ RACE) revealed asloRtranscription start site in the IGR, from which the −10 and −35 promoter regions were predicted. The results of gel mobility shift assays support direct SloR binding to the IGR, albeit with a lower affinity than that for SloR binding to thesloABCRpromoter. The function of thesloRpromoter was validated by semiquantitative real-time PCR (qRT-PCR) experiments. Interestingly,sloRexpression was not significantly affected when bacteria were grown in the presence of a high manganese concentration, whereas expression of thesloABCoperon was repressed under these conditions. The results ofin vitrotranscription studies support the occurrence of SloR-mediated transcriptional activation ofsloRand repression ofsloABC. Taken together, these findings implicate SloR as a bifunctional regulator that repressessloABCpromoter activity and encouragessloRtranscription from an independent promoter.IMPORTANCETooth decay is a ubiquitous infectious disease that is especially pervasive in underserved communities worldwide.S. mutans-induced carious lesions cause functional, physical, and/or esthetic impairment in the vast majority of adults and in 60 to 90% of schoolchildren in industrialized countries. Billions of dollars are spent annually on caries treatment, and productivity losses due to absenteeism from the workplace are significant. Research aimed at alleviatingS. mutans-induced tooth decay is important because it can address the socioeconomic disparity that is associated with dental cavities and improve overall general health, which is inextricably linked to oral health. Research focused on theS. mutansSloR metalloregulatory protein can guide the development of novel therapeutics and thus alleviate the burden of dental cavities.

scholarly journals Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
A. Sukumaran ◽  
S. Pladwig ◽  
J. Geddes-McAlister
Keyword(s):  
Klebsiella Pneumoniae ◽  
Metal Ion ◽  
Ion Homeostasis ◽  
Cellular Protein ◽  
Pcr Analysis ◽  
Phenotypic Analysis ◽  
Metal Ion Homeostasis ◽  
Capsule Production ◽  
The Impact

Abstract Background Microbial organisms encounter a variety of environmental conditions, including changes to metal ion availability. Metal ions play an important role in many biological processes for growth and survival. As such, microbes alter their cellular protein levels and secretion patterns in adaptation to a changing environment. This study focuses on Klebsiella pneumoniae, an opportunistic bacterium responsible for nosocomial infections. By using K. pneumoniae, we aim to determine how a nutrient-limited environment (e.g., zinc depletion) modulates the cellular proteome and secretome of the bacterium. By testing virulence in vitro, we provide novel insight into bacterial responses to limited environments in the presence of the host. Results Analysis of intra- and extracellular changes identified 2380 proteins from the total cellular proteome (cell pellet) and 246 secreted proteins (supernatant). Specifically, HutC, a repressor of the histidine utilization operon, showed significantly increased abundance under zinc-replete conditions, which coincided with an expected reduction in expression of genes within the hut operon from our validating qRT-PCR analysis. Additionally, we characterized a putative cation transport regulator, ChaB that showed significantly higher abundance under zinc-replete vs. -limited conditions, suggesting a role in metal ion homeostasis. Phenotypic analysis of a chaB deletion strain demonstrated a reduction in capsule production, zinc-dependent growth and ion utilization, and reduced virulence when compared to the wild-type strain. Conclusions This is first study to comprehensively profile the impact of zinc availability on the proteome and secretome of K. pneumoniae and uncover a novel connection between zinc transport and capsule production in the bacterial system.

scholarly journals Mechanism of metal ion-induced activation of a two-component sensor kinase

2019 ◽  
Vol 476 (1) ◽  
pp. 115-135 ◽  
Author(s):  
Trisiani Affandi ◽  
Megan M. McEvoy
Keyword(s):  
Metal Ion ◽  
Ion Homeostasis ◽  
Cytoplasmic Domain ◽  
Metal Resistance ◽  
Phosphoryl Group ◽  
Metal Ion Binding ◽  
Metal Ion Homeostasis ◽  
Two Component

Abstract Two-component systems (TCSs) are essential for bacteria to sense, respond, and adapt to changing environments, such as elevation of Cu(I)/Ag(I) ions in the periplasm. In Escherichia coli, the CusS–CusR TCS up-regulates the cusCFBA genes under increased periplasmic Cu(I)/Ag(I) concentrations to help maintain metal ion homeostasis. The CusS histidine kinase is a homodimeric integral membrane protein that binds to periplasmic Cu(I)/Ag(I) and transduces a signal to its cytoplasmic kinase domain. However, the mechanism of how metal binding in the periplasm activates autophosphorylation in the cytoplasm is unknown. Here, we report that only one of the two metal ion-binding sites in CusS enhances dimerization of the sensor domain. Utilizing nanodisc technology to study full-length CusS, we show that metal-induced dimerization in the sensor domain triggers kinase activity in the cytoplasmic domain. We also investigated autophosphorylation in the cytoplasmic domain of CusS and phosphotransfer between CusS and CusR. In vitro analyses show that CusS autophosphorylates its conserved H271 residue at the N1 position of the histidine imidazole. The phosphoryl group is removed by the response regulator CusR in a reaction that requires a conserved aspartate at position 51. Functional analyses in vivo of CusS and CusR variants with mutations in the autophosphorylation or phosphoacceptor residues suggest that the phosphotransfer event is essential for metal resistance in E. coli. Biochemical analysis shows that the CusS dimer autophosphorylates using a cis mechanism. Our results support a signal transduction model in which rotation and bending movements in the cytoplasmic domain maintain the mode of autophosphorylation.

scholarly journals Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production

2021 ◽  
Author(s):  
Arjun Sukumaran ◽  
Samanta Pladwig ◽  
Jennifer Geddes-McAlister
Keyword(s):  
Klebsiella Pneumoniae ◽  
Metal Ion ◽  
Ion Homeostasis ◽  
Cellular Protein ◽  
Pcr Analysis ◽  
Phenotypic Analysis ◽  
Metal Ion Homeostasis ◽  
Capsule Production ◽  
The Impact

Abstract Background: Microbial organisms encounter a variety of environmental conditions, including changes to metal ion availability. Metal ions play an important role in many biological processes for growth and survival. As such, microbes alter their cellular protein levels and secretion patterns in adaptation to a changing environment. This study focuses on Klebsiella pneumoniae, an opportunistic bacterium responsible for nosocomial infections. By using K. pneumoniae, we aim to determine how a nutrient-limited environment (e.g., zinc depletion) modulates the cellular proteome and secretome of the bacterium. By testing virulence in vitro, we provide novel insight into bacterial responses to limited environments in the presence of the host. Results: Analysis of intra- and extracellular changes identified 2,380 proteins from the total cellular proteome (cell pellet) and 246 secreted proteins (supernatant). Specifically, HutC, a repressor of the histidine utilization operon, showed significantly increased abundance under zinc-replete conditions, which coincided with an expected reduction in expression of genes within the hut operon from our validating qRT-PCR analysis. Additionally, we characterized a putative cation transport regulator, ChaB that showed significantly higher abundance under zinc-replete vs. -limited conditions, suggesting a role in metal ion homeostasis. Phenotypic analysis of a chaB deletion strain demonstrated a reduction in capsule production, zinc-dependent growth and ion utilization, and reduced virulence when compared to the wild-type strain. Conclusions: This is first study to comprehensively profile the impact of zinc availability on the proteome and secretome of K. pneumoniae and uncover a novel connection between zinc transport and capsule production in the bacterial system.

Structural analysis and insight into metal-ion activation of the iron-dependent regulator fromThermoplasma acidophilum

2014 ◽  
Vol 70 (5) ◽  
pp. 1281-1288 ◽  
Author(s):  
Hyun Ku Yeo ◽  
Young Woo Park ◽  
Jae Young Lee
Keyword(s):  
Binding Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Ion Homeostasis ◽  
Binding Property ◽  
Metal Ion Binding ◽  
Mobility Shift ◽  
Iron Cluster ◽  
Metal Binding Site ◽  
Metal Ion Homeostasis

The iron-dependent regulator (IdeR) is a metal ion-activated transcriptional repressor that regulates the expression of genes encoding proteins involved in iron uptake to maintain metal-ion homeostasis. IdeR is a functional homologue of the diphtheria toxin repressor (DtxR), and both belong to the DtxR/MntR family of metalloregulators. The structure of Fe2+-bound IdeR (TA0872) fromThemoplasma acidophilumwas determined at 2.1 Å resolution by X-ray crystallography using single-wavelength anomalous diffraction. The presence of Fe2+, which is the true biological activator of IdeR, in the metal-binding site was ascertained by the use of anomalous difference electron-density maps using diffraction data collected at the Fe absorption edge. Each DtxR/IdeR subunit contains two metal ion-binding sites separated by 9 Å, labelled the primary and ancillary sites, whereas the crystal structures of IdeR fromT. acidophilumshow a binuclear iron cluster separated by 3.2 Å, which is novel toT. acidophilumIdeR. The metal-binding site analogous to the primary site in DtxR was unoccupied, and the ancillary site was occupied by binuclear clustered ions. This difference suggests thatT. acidophilumIdeR and its closely related homologues are regulated by a mechanism distinct from that of either DtxR or MntR.T. acidophilumIdeR was also shown to have a metal-dependent DNA-binding property by electrophoretic mobility shift assay.

scholarly journals The First Histidine Triad Motif of PhtD Is Critical for Zinc Homeostasis inStreptococcus pneumoniae

2015 ◽  
Vol 84 (2) ◽  
pp. 407-415 ◽  
Author(s):  
Bart A. Eijkelkamp ◽  
Victoria G. Pederick ◽  
Charles D. Plumptre ◽  
Richard M. Harvey ◽  
Catherine E. Hughes ◽  
...  
Keyword(s):  
Metal Ion ◽  
Ion Homeostasis ◽  
Zinc Homeostasis ◽  
Zinc Binding ◽  
Content Type ◽  
Metal Ion Homeostasis ◽  
Expression Studies ◽  
The Individual

Streptococcus pneumoniaeis the world's foremost human pathogen. Acquisition of the first row transition metal ion zinc is essential for pneumococcal colonization and disease. Zinc is acquired via the ATP-binding cassette transporter AdcCB and two zinc-binding proteins, AdcA and AdcAII. We have previously shown that AdcAII is reliant upon the polyhistidine triad (Pht) proteins to aid in zinc recruitment. Pht proteins generally contain five histidine (His) triad motifs that are believed to facilitate zinc binding and therefore play a significant role in pneumococcal metal ion homeostasis. However, the importance and potential redundancy of these motifs have not been addressed. We examined the effects of mutating each of the five His triad motifs of PhtD. The combination ofin vitrogrowth assays, active zinc uptake, and PhtD expression studies show that the His triad closest to the protein's amino terminus is the most important for zinc acquisition. Intriguingly,in vivocompetitive infection studies investigating the amino- and carboxyl-terminal His triad mutants indicate that the motifs have similar importance in colonization. Collectively, our new insights into the contributions of the individual His triad motifs of PhtD, and by extension the other Pht proteins, highlight the crucial role of the first His triad site in zinc acquisition. This study also suggests that the Pht proteins likely play a role beyond zinc acquisition in pneumococcal virulence.

scholarly journals Metallothionein: A Multifunctional Protein from Toxicity to Cancer

2003 ◽  
Vol 18 (3) ◽  
pp. 162-169 ◽  
Author(s):  
S.E. Theocharis ◽  
A.P. Margeli ◽  
A. Koutselinis
Keyword(s):  
Cell Proliferation ◽  
Metal Ion ◽  
Defense Mechanisms ◽  
Ion Homeostasis ◽  
Low Molecular Weight ◽  
Multifunctional Protein ◽  
Metal Ion Homeostasis ◽  
Potential Marker ◽  
Proliferation And Apoptosis ◽  
Carcinogenic Process

The metallothionein (MT) family is a class of low molecular weight, intracellular and cysteine-rich proteins presenting high affinity for metal ions. Although the members of this family were discovered nearly 40 years ago, their functional significance remains obscure. Four major MT isoforms, MT-1, MT-2, MT-3 and MT-4, have been identified in mammals. MTs are involved in many pathophysiological processes such as metal ion homeostasis and detoxification, protection against oxidative damage, cell proliferation and apoptosis, chemoresistance and radiotherapy resistance. MT isoforms have been shown to be involved in several aspects of the carcinogenic process, cancer development and progression. MT expression has been implicated as a transient response to any form of stress or injury providing cytoprotective action. Although MT participates in the carcinogenic process, its use as a potential marker of tumor differentiation or cell proliferation, or as a predictor of poor prognosis remains unclear. In the present review the involvement of MT in defense mechanisms to toxicity and in carcinogenicity is discussed.

scholarly journals The novel ECF56 SigG1-RsfG system modulates morphological differentiation and metal-ion homeostasis in Streptomyces tsukubaensis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Rute Oliveira ◽  
Matthew J. Bush ◽  
Sílvia Pires ◽  
Govind Chandra ◽  
Delia Casas-Pastor ◽  
...  
Keyword(s):  
Metal Ion ◽  
Ion Homeostasis ◽  
Morphological Differentiation ◽  
The Novel ◽  
Copper Trafficking ◽  
Streptomyces Tsukubaensis ◽  
Metal Ion Homeostasis ◽  
Σ Factor ◽  
Domain Organisation

AbstractExtracytoplasmic function (ECF) sigma factors are key transcriptional regulators that prokaryotes have evolved to respond to environmental challenges. Streptomyces tsukubaensis harbours 42 ECFs to reprogram stress-responsive gene expression. Among them, SigG1 features a minimal conserved ECF σ2–σ4 architecture and an additional C-terminal extension that encodes a SnoaL_2 domain, which is characteristic for ECF σ factors of group ECF56. Although proteins with such domain organisation are widely found among Actinobacteria, the functional role of ECFs with a fused SnoaL_2 domain remains unknown. Our results show that in addition to predicted self-regulatory intramolecular amino acid interactions between the SnoaL_2 domain and the ECF core, SigG1 activity is controlled by the cognate anti-sigma protein RsfG, encoded by a co-transcribed sigG1-neighbouring gene. Characterisation of ∆sigG1 and ∆rsfG strains combined with RNA-seq and ChIP-seq experiments, suggests the involvement of SigG1 in the morphological differentiation programme of S. tsukubaensis. SigG1 regulates the expression of alanine dehydrogenase, ald and the WhiB-like regulator, wblC required for differentiation, in addition to iron and copper trafficking systems. Overall, our work establishes a model in which the activity of a σ factor of group ECF56, regulates morphogenesis and metal-ions homeostasis during development to ensure the timely progression of multicellular differentiation.

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