scholarly journals RR06 Activates Transcription of spr1996 and cbpA in Streptococcus pneumoniae

2007 ◽  
Vol 189 (6) ◽  
pp. 2497-2509 ◽  
Author(s):  
Zhuo Ma ◽  
Jing-Ren Zhang
Keyword(s):  
Streptococcus Pneumoniae ◽  
Transcriptional Activation ◽  
Protective Antigen ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Promoter Regions ◽  
Conserved Sequence ◽  
Antibody Staining ◽  
Regulator Protein ◽  
Bacterial Replication

ABSTRACT Streptococcus pneumoniae colonizes at the nasopharynx of humans and is able to disseminate and cause various infections. The hallmark of pneumococcal disease is rapid bacterial replication in different tissue sites leading to intense inflammation. The genetic basis of pneumococcal adaptation to different host niches remains sketchy. In this study, we investigated the regulatory effect of RR06, a response regulator protein, on gene expression of S. pneumoniae. Microarray and Northern blot analyses showed that RR06 is specifically required for transcription of spr1996 and cbpA. While the function of Spr1996 is unknown, CbpA has been well characterized as a surface-exposed protective antigen and a virulence factor of S. pneumoniae. A recombinant form of RR06 was able to bind to a 19-bp conserved sequence shared by the spr1996 and cbpA promoter regions. Furthermore, inactivation of rr06 resulted in loss of CbpA expression as detected by antibody staining and bacterial adhesion. CbpA expression was restored in trans by the intact rr06 gene. However, a mutant, RR06(D51A), with a point mutation in the aspartate residue at position 51 (a predicted major phosphorylation site) of RR06, completely abolished the CbpA expression, suggesting that RR06 phosphorylation is required for transcriptional activation of spr1996 and cbpA. Finally, inactivation of rr06 in additional pneumococcal strains also led to the loss of CbpA expression. These data implicate that RR06 activates the expression of spr1996 and cbpA in many other pneumococcal strains.

scholarly journals Switched or Not?: the Structure of Unphosphorylated CheY Bound to the N Terminus of FliM

2006 ◽  
Vol 188 (21) ◽  
pp. 7354-7363 ◽  
Author(s):  
Collin M. Dyer ◽  
Frederick W. Dahlquist
Keyword(s):  
Binding Site ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Active Role ◽  
Crystallographic Structure ◽  
Coupling Mechanism ◽  
Allosteric Communication ◽  
Regulator Protein ◽  
Target Binding ◽  
Response Regulator Protein

ABSTRACT Phosphorylation of Escherichia coli CheY increases its affinity for its target, FliM, 20-fold. The interaction between BeF3 −-CheY, a phosphorylated CheY (CheY∼P) analog, and the FliM sequence that it binds has been described previously in molecular detail. Although the conformation that unphosphorylated CheY adopts in complex with FliM was unknown, some evidence suggested that it is similar to that of CheY∼P. To resolve the issue, we have solved the crystallographic structure of unphosphorylated, magnesium(II)-bound CheY in complex with a synthetic peptide corresponding to the target region of FliM (the 16 N-terminal residues of FliM [FliM16]). While the peptide conformation and binding site are similar to those of the BeF3 −-CheY-FliM16 complex, the inactive CheY conformation is largely retained in the unphosphorylated Mg2+-CheY-FliM16 complex. Communication between the target binding site and the phosphorylation site, observed previously in biochemical experiments, is enabled by a network of conserved side chain interactions that partially mimic those observed in BeF3 −-activated CheY. This structure makes clear the active role that the β4-α4 loop plays in the Tyr87-Tyr106 coupling mechanism that enables allosteric communication between the phosphorylation site and the target binding surface. Additionally, this structure provides a high-resolution view of an intermediate conformation of a response regulator protein, which had been generally assumed to be two state.

scholarly journals An Unstable Competence-Induced Protein, CoiA, Promotes Processing of Donor DNA after Uptake during Genetic Transformation in Streptococcus pneumoniae

2006 ◽  
Vol 188 (14) ◽  
pp. 5177-5186 ◽  
Author(s):  
Bhushan V. Desai ◽  
Donald A. Morrison
Keyword(s):  
Streptococcus Pneumoniae ◽  
Genetic Transformation ◽  
Transcriptional Activation ◽  
Response Regulator ◽  
Specific Response ◽  
Dna Uptake ◽  
Gram Positive Bacteria ◽  
Specific Alternative ◽  
Natural Genetic Transformation

ABSTRACT Natural genetic transformation in Streptococcus pneumoniae entails transcriptional activation of at least two sets of genes. One set of genes, activated by the competence-specific response regulator ComE, is involved in initiating competence, whereas a second set is activated by the competence-specific alternative sigma factor ComX and functions in DNA uptake and recombination. Here we report an initial characterization of CoiA, a ComX-dependent gene product that is induced during competence and is required for transformation. CoiA is widely conserved among gram-positive bacteria, and in streptococci, the entire coiA locus composed of four genes is conserved. By use of immunoblot assay, we show that, similar to its message, CoiA protein is transient, appearing at 10 min and largely disappearing by 30 min post-competence induction. Using complementation analysis, we establish that coiA is the only gene of this induced locus needed for transformability. We find no indication of CoiA having a role in regulating competence. Finally, using 32P- and 3H-labeled donor DNA, we demonstrate that a coiA mutant can internalize normal amounts of donor DNA compared to the wild-type strain but is unable to process it into viable transformants, suggesting a role for CoiA after DNA uptake, either in DNA processing or recombination.

Dual phosphorylation in response regulator protein PrrA is crucial for intracellular survival of mycobacteria consequent upon transcriptional activation

2017 ◽  
Vol 474 (24) ◽  
pp. 4119-4136 ◽  
Author(s):  
Alok K. Mishra ◽  
Shivraj M. Yabaji ◽  
Rikesh K. Dubey ◽  
Ekta Dhamija ◽  
Kishore K. Srivastava
Keyword(s):  
Transcriptional Activation ◽  
Response Regulator ◽  
Regulatory Protein ◽  
Peritoneal Macrophages ◽  
Physical Interaction ◽  
Fast Growing ◽  
Regulatory Circuit ◽  
Regulator Protein ◽  
Promoter Dna

The remarkable ability of Mycobacterium tuberculosis (Mtb) to survive inside human macrophages is attributed to the presence of a complex sensory and regulatory network. PrrA is a DNA-binding regulatory protein, belonging to an essential two-component system (TCS), PrrA/B, which is required for early phase intracellular replication of Mtb. Despite its importance, the mechanism of PrrA/B-mediated signaling is not well understood. In the present study, we demonstrate that the binding of PrrA on the promoter DNA and its consequent activation is cumulatively controlled via dual phosphorylation of the protein. We have further characterized the role of terminal phospho-acceptor domain in the physical interaction of PrrA with its cognate kinase PrrB. The genetic deletion of prrA/B in Mycobacterium smegmatis was possible only in the presence of ectopic copies of the genes, suggesting the essentiality of this TCS in fast-growing mycobacterial strains as well. The overexpression of phospho-mimetic mutant (T6D) altered the growth of M. smegmatis in an in vitro culture and affected the replication of Mycobacterium bovis BCG in mouse peritoneal macrophages. Interestingly, the Thr6 site was found to be conserved in Mtb complex, whereas it was altered in some fast-growing mycobacterial strains, indicating that this unique phosphorylation might be predominant in employing the regulatory circuit in M. bovis BCG and presumably also in Mtb complex.

scholarly journals Transcriptional Activation by Bacillus subtilis ResD: Tandem Binding to Target Elements and Phosphorylation-Dependent and -Independent Transcriptional Activation

2004 ◽  
Vol 186 (7) ◽  
pp. 2028-2037 ◽  
Author(s):  
Hao Geng ◽  
Shunji Nakano ◽  
Michiko M. Nakano
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Activation ◽  
Response Regulator ◽  
Gene Activation ◽  
Phosphorylation Site ◽  
Oxygen Limitation ◽  
Nitrate Respiration ◽  
Expression Of Genes

ABSTRACT The expression of genes involved in nitrate respiration in Bacillus subtilis is regulated by the ResD-ResE two-component signal transduction system. The membrane-bound ResE sensor kinase perceives a redox-related signal(s) and phosphorylates the cognate response regulator ResD, which enables interaction of ResD with ResD-dependent promoters to activate transcription. Hydroxyl radical footprinting analysis revealed that ResD tandemly binds to the −41 to −83 region of hmp and the −46 to −92 region of nasD. In vitro runoff transcription experiments showed that ResD is necessary and sufficient to activate transcription of the ResDE regulon. Although phosphorylation of ResD by ResE kinase greatly stimulated transcription, unphosphorylated ResD, as well as ResD with a phosphorylation site (Asp57) mutation, was able to activate transcription at a low level. The D57A mutant was shown to retain the activity in vivo to induce transcription of the ResDE regulon in response to oxygen limitation, suggesting that ResD itself, in addition to its activation through phosphorylation-mediated conformation change, senses oxygen limitation via an unknown mechanism leading to anaerobic gene activation.

scholarly journals Control of acetyl phosphate-dependent phosphorylation of the response regulator CiaR by acetate kinase in Streptococcus pneumoniae

Microbiology ◽  
2020 ◽  
Vol 166 (4) ◽  
pp. 411-421
Author(s):  
Sabrina Kaiser ◽  
Lisa Marie Hoppstädter ◽  
Kevser Bilici ◽  
Kevin Heieck ◽  
Reinhold Brückner
Keyword(s):  
Streptococcus Pneumoniae ◽  
Transcriptional Activation ◽  
Type Species ◽  
Response Regulator ◽  
Acetate Kinase ◽  
Acetyl Phosphate ◽  
Content Type ◽  
Link Type ◽  
Cell Extracts

The two-component regulatory system CiaRH of Streptococcus pneumoniae affects a large variety of physiological processes including ß-lactam resistance, competence development, maintenance of cell integrity, bacteriocin production, but also host colonization and virulence. The response regulator CiaR is active under a wide variety of conditions and the cognate CiaH kinase is not always needed to maintain CiaR activity. Using tetracycline-controlled expression of ciaR and variants, acetyl phosphate was identified in vivo as the alternative source of CiaR phosphorylation in the absence of CiaH. Concomitant inactivation of ciaH and the acetate kinase gene ackA led to very high levels of CiaR-mediated promoter activation. Strong transcriptional activation was accompanied by a high phosphorylation status of CiaR as determined by Phos-tag gel electrophoresis of S. pneumoniae cell extracts. Furthermore, AckA acted negatively upon acetyl phosphate-dependent phosphorylation of CiaR. Experiments using the Escherichia coli two-hybrid system based on adenylate cyclase reconstitution indicated binding of AckA to CiaR and therefore direct regulation. Subsequent in vitro CiaR phosphorylation experiments confirmed in vivo observations. Purified AckA was able to inhibit acetyl phosphate-dependent phosphorylation. Inhibition required the presence of ADP. AckA-mediated regulation of CiaR phosphorylation is the first example for a regulatory connection of acetate kinase to a response regulator besides controlling acetyl phosphate levels. It will be interesting to see if this novel regulation applies to other response regulators in S. pneumoniae or even in other organisms.

scholarly journals Microarray-Based Identification of a NovelStreptococcus pneumoniae Regulon Controlled by an Autoinduced Peptide

2000 ◽  
Vol 182 (17) ◽  
pp. 4696-4703 ◽  
Author(s):  
Antoine de Saizieu ◽  
Christophe Gardès ◽  
Nicholas Flint ◽  
Christian Wagner ◽  
Markus Kamber ◽  
...  
Keyword(s):  
Response Regulator ◽  
Open Reading Frames ◽  
Leader Peptide ◽  
Recognition Sequence ◽  
Promoter Regions ◽  
Sequence Element ◽  
Conserved Sequence ◽  
Insertional Inactivation ◽  
Genes Encoding ◽  
Processed Form

ABSTRACT We have identified in the Streptococcus pneumoniaegenome sequence a two-component system (TCS13, Blp [bacteriocin-like peptide]) which is closely related to quorum-sensing systems regulating cell density-dependent phenotypes such as the development of genetic competence or the production of antimicrobial peptides in lactic acid bacteria. In this study we present evidence that TCS13 is a peptide-sensing system that controls a regulon including genes encoding Blps. Downstream of the Blp TCS (BlpH R) we identified open reading frames (blpAB) that have the potential to encode an ABC transporter that is homologous to the ComA/B export system for the competence-stimulating peptide ComC. The putative translation product of blpC, a small gene located downstream ofblpAB, has a leader peptide with a Gly-Gly motif. This leader peptide is typical of precursors processed by this family of transporters. Microarray-based expression profiling showed that a synthetic oligopeptide corresponding to the processed form of BlpC (BlpC*) induces a distinct set of 16 genes. The changes in the expression profile elicited by synthetic BlpC* depend on BlpH since insertional inactivation of its corresponding gene abolishes differential gene induction. Comparison of the promoter regions of theblp genes disclosed a conserved sequence element formed by two imperfect direct repeats upstream of extended −10 promoter elements. We propose that BlpH is the sensor for BlpC* and the conserved sequence element is a recognition sequence for the BlpR response regulator.

scholarly journals EGFR-ERK induced activation of GRHL1 promotes cell cycle progression by up-regulating cell cycle related genes in lung cancer

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Yiming He ◽  
Mingxi Gan ◽  
Yanan Wang ◽  
Tong Huang ◽  
Jianbin Wang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Potential Role ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Phosphorylation Site ◽  
Promoter Regions ◽  
Cycle Progression

AbstractGrainyhead-like 1 (GRHL1) is a transcription factor involved in embryonic development. However, little is known about the biological functions of GRHL1 in cancer. In this study, we found that GRHL1 was upregulated in non-small cell lung cancer (NSCLC) and correlated with poor survival of patients. GRHL1 overexpression promoted the proliferation of NSCLC cells and knocking down GRHL1 inhibited the proliferation. RNA sequencing showed that a series of cell cycle-related genes were altered when knocking down GRHL1. We further demonstrated that GRHL1 could regulate the expression of cell cycle-related genes by binding to the promoter regions and increasing the transcription of the target genes. Besides, we also found that EGF stimulation could activate GRHL1 and promoted its nuclear translocation. We identified the key phosphorylation site at Ser76 on GRHL1 that is regulated by the EGFR-ERK axis. Taken together, these findings elucidate a new function of GRHL1 on regulating the cell cycle progression and point out the potential role of GRHL1 as a drug target in NSCLC.

scholarly journals Glucocorticoid Receptor Phosphorylation Differentially Affects Target Gene Expression

2008 ◽  
Vol 22 (8) ◽  
pp. 1754-1766 ◽  
Author(s):  
Weiwei Chen ◽  
Thoa Dang ◽  
Raymond D. Blind ◽  
Zhen Wang ◽  
Claudio N. Cavasotto ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Target Genes ◽  
Divalent Cations ◽  
Phosphorylation Site ◽  
Transcriptional Response ◽  
Receptor Occupancy ◽  
Wild Type ◽  
Interacting Protein

Abstract The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

scholarly journals The Two-Component Regulatory System TCS08 Is Involved in Cellobiose Metabolism of Streptococcus pneumoniae R6

2006 ◽  
Vol 189 (4) ◽  
pp. 1342-1350 ◽  
Author(s):  
Stuart J. McKessar ◽  
Regine Hakenbeck
Keyword(s):  
Streptococcus Pneumoniae ◽  
Response Regulator ◽  
Regulatory System ◽  
Phosphotransferase System ◽  
Two Component System ◽  
Transcription Profiles ◽  
Regulatory Systems ◽  
Two Component ◽  
Cognate Response Regulator ◽  
Gram Positive Cocci

ABSTRACT The two-component system TCS08 is one of the regulatory systems that is important for virulence of Streptococcus pneumoniae. In order to investigate the TCS08 regulon, we have analyzed transcription profiles of mutants derived from S. pneumoniae R6 by microarray analysis. Since deletion mutants are often without a significant phenotype, we constructed a mutation in the histidine kinase HK08, T133P, in analogy to the phosphatase mutation T230P in the H box of the S. pneumoniae CiaH kinase described recently (D. Zähner, K. Kaminski, M. van der Linden, T. Mascher, M. Merai, and R. Hakenbeck, J. Mol. Microbiol. Biotechnol. 4:211-216, 2002). In addition, a deletion mutation was constructed in rr08, encoding the cognate response regulator. The most heavily suppressed genes in the hk08 mutant were spr0276 to spr0282, encoding a putative cellobiose phosphoenolpyruvate sugar phosphotransferase system (PTS). Whereas the R6 Smr parent strain and the Δrr08 mutant readily grew on cellobiose, the hk08 mutant and selected mutants with deletions in the PTS cluster did not, strongly suggesting that TCS08 is involved in the catabolism of cellobiose. Homologues of the TCS08 system were found in closely related streptococci and other gram-positive cocci. However, the genes spr0276 to spr0282, encoding the putative cellobiose PTS, represent a genomic island in S. pneumoniae and homologues were found in Streptococcus gordonii only, suggesting that this system might contribute to the pathogenicity potential of the pneumococcus.

Identification of an upstream regulatory sequence that mediates the transcription of mox genes in Methylobacterium extorquens AM1

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3723-3728 ◽  
Author(s):  
Meng Zhang ◽  
Kelly A. FitzGerald ◽  
Mary E. Lidstrom
Keyword(s):  
Promoter Activity ◽  
Site Directed Mutagenesis ◽  
Regulatory Sequence ◽  
Methylobacterium Extorquens ◽  
Promoter Regions ◽  
Enhancer Element ◽  
Conserved Sequence ◽  
Normal Expression ◽  
Genetic Constructs ◽  
Transcriptional Fusions

A multiple A-tract sequence has been identified in the promoter regions for the mxaF, pqqA, mxaW, mxbD and mxcQ genes involved in methanol oxidation in Methylobacterium extorquens AM1, a facultative methylotroph. Site-directed mutagenesis was exploited to delete or change this conserved sequence. Promoter-xylE transcriptional fusions were used to assess promoter activity in these mutants. A fiftyfold drop in the XylE activity was observed for the mxaF and pqqA promoters without this sequence, and a five- to sixfold drop in the XylE activity was observed for the mxbD and mxcQ promoters without this sequence. Mutants were generated in the chromosomal copies in which this sequence was either deleted or altered, and these mutants were unable to grow on methanol. When one of these sequences was added to Plac of Escherichia coli, which is a weak constitutive promoter in M. extorquens AM1, the activity increased two- to threefold. These results suggest that this sequence is essential for normal expression of these genes in M. extorquens AM1, and may serve as a general enhancer element for genetic constructs in this bacterium.

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