scholarly journals Autoregulation of Lantibiotic Bovicin HJ50 Biosynthesis by the BovK-BovR Two-Component Signal Transduction System inStreptococcus bovisHJ50

2010 ◽  
Vol 77 (2) ◽  
pp. 407-415 ◽  
Author(s):  
Jianqiang Ni ◽  
Kunling Teng ◽  
Gang Liu ◽  
Caixia Qiao ◽  
Liandong Huan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Fluorescent Protein ◽  
Specific Binding ◽  
Biosynthetic Gene Cluster ◽  
Phosphoryl Group ◽  
Signal Transduction System ◽  
Two Component ◽  
Two Component Signal Transduction ◽  
Green Fluorescent

ABSTRACTStreptococcus bovisHJ50 produces a lacticin 481-like 33-amino-acid-residue lantibiotic, designated bovicin HJ50.bovK-bovRin the bovicin HJ50 biosynthetic gene cluster is predicted to be a two-component signal transduction system involved in sensing signals and regulating gene expression. Disruption ofbovKorbovRresulted in the abrogation of bovicin HJ50 production, suggesting both genes play important roles in bovicin HJ50 biosynthesis. Addition of exogenous bovicin HJ50 peptide to cultures of abovMmutant that lost the capability for bovicin HJ50 production and structural genebovAtranscription inS. bovisHJ50 induced dose-dependent transcription of thebovAgene, demonstrating that bovicin HJ50 production was normally autoregulated. The transcription ofbovAwas no longer induced by bovicin HJ50 inbovKandbovRdisruption mutants, suggesting that BovK-BovR plays an essential role in the signal transduction regulating bovicin HJ50 biosynthesis. A phosphorylation assay indicated that BovK has the ability to autophosphorylate and subsequently transfer the phosphoryl group to the downstream BovR protein to carry on signal transduction. Electromobility shift assays (EMSA) and green fluorescent protein (GFP) reporter gene expression assays showed the specific binding of BovR to thebovApromoter, indicating that BovR regulatesbovAexpression by direct binding between them. Taken together, bovicin HJ50 biosynthesis is induced by bovicin HJ50 itself and regulated via the two-component signal transduction system BovK-BovR.

scholarly journals Development and Validation of a High-Throughput Cell-Based Screen To Identify Activators of a Bacterial Two-Component Signal Transduction System

2015 ◽  
Vol 59 (7) ◽  
pp. 3789-3799 ◽  
Author(s):  
Julia J. van Rensburg ◽  
Kate R. Fortney ◽  
Lan Chen ◽  
Andrew J. Krieger ◽  
Bruno P. Lima ◽  
...  
Keyword(s):  
Signal Transduction ◽  
High Throughput ◽  
Response Regulator ◽  
Phosphoryl Group ◽  
Signal Transduction System ◽  
Membrane Repair ◽  
Content Type ◽  
Two Component ◽  
Serovar Typhimurium ◽  
Two Component Signal Transduction

ABSTRACTCpxRA is a two-component signal transduction system (2CSTS) found in many drug-resistant Gram-negative bacteria. In response to periplasmic stress, CpxA autophosphorylates and donates a phosphoryl group to its cognate response regulator, CpxR. Phosphorylated CpxR (CpxR-P) upregulates genes involved in membrane repair and downregulates multiple genes that encode virulence factors, which are trafficked across the cell membrane. Mutants that constitutively activate CpxRA inSalmonella entericaserovar Typhimurium andHaemophilus ducreyiare avirulent in mice and humans, respectively. Thus, the activation of CpxRA has high potential as a novel antimicrobial/antivirulence strategy. Using a series ofEscherichia colistrains containing a CpxR-P-responsivelacZreporter and deletions in genes encoding CpxRA system components, we developed and validated a novel cell-based high-throughput screen (HTS) for CpxRA activators. A screen of 36,000 compounds yielded one hit compound that increased reporter activity in wild-type cells. This is the first report of a compound that activates, rather than inhibits, a 2CSTS. The activity profile of the compound against CpxRA pathway mutants in the presence of glucose suggested that the compound inhibits CpxA phosphatase activity. We confirmed that the compound induced the accumulation of CpxR-P in treated cells. Although the hit compound contained a nitro group, a derivative lacking this group retained activity in serum and had lower cytotoxicity than that of the initial hit. This HTS is amenable for the screening of larger libraries to find compounds that activate CpxRA by other mechanisms, and it could be adapted to find activators of other two-component systems.

scholarly journals Identification of a Two-Component Signal Transduction System fromCorynebacterium diphtheriae That Activates Gene Expression in Response to the Presence of Heme and Hemoglobin

1999 ◽  
Vol 181 (17) ◽  
pp. 5330-5340 ◽  
Author(s):  
Michael P. Schmitt
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Response Regulator ◽  
Open Reading Frames ◽  
Sensor Kinase ◽  
Dependent Manner ◽  
Signal Transduction System ◽  
Kinase Gene ◽  
Two Component ◽  
Two Component Signal Transduction

ABSTRACT Corynebacterium diphtheriae, the causative agent of diphtheria, utilizes various host compounds to acquire iron. TheC. diphtheriae hmuO gene encodes a heme oxygenase that is involved in the utilization of heme and hemoglobin as iron sources. Transcription of the hmuO gene in C. diphtheriae is controlled under a dual regulatory mechanism in which the diphtheria toxin repressor protein (DtxR) and iron repress expression while either heme or hemoglobin is needed to activate transcription. In this study, two clones isolated from a C. diphtheriae chromosomal library were shown to activate transcription from the hmuO promoter in Escherichia coli. Sequence analysis revealed that these activator clones each carried distinct genes whose products had significant homology to response regulators of two-component signal transduction systems. Located upstream from each of these response regulator homologs are partial open reading frames that are predicted to encode the C-terminal portions of sensor kinases. The full-length sensor kinase gene for each of these systems was cloned from the C. diphtheriaechromosome, and constructs each carrying one complete sensor kinase gene and its cognate response regulator were constructed. One of these constructs, pTSB20, which carried the response regulator (chrA) and its cognate sensor kinase (chrS), was shown to strongly activate transcription from the hmuOpromoter in a heme-dependent manner in E. coli. A mutation in chrA (chrAD50N), which changed a conserved aspartic acid residue at position 50, the presumed site of phosphorylation by ChrS, to an asparagine, abolished heme-dependent activation. These findings suggest that the sensor kinase ChrS is involved in the detection of heme and the transduction of this signal, via a phosphotransfer mechanism, to the response regulator ChrA, which then activates transcription of the hmuO promoter. This is the first report of a bacterial two-component signal transduction system that controls gene expression through a heme-responsive mechanism.

Architecture of the complete oxygen-sensing FixL-FixJ two-component signal transduction system

2018 ◽  
Vol 11 (525) ◽  
pp. eaaq0825 ◽  
Author(s):  
Gareth S. A. Wright ◽  
Akane Saeki ◽  
Takaaki Hikima ◽  
Yoko Nishizono ◽  
Tamao Hisano ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Oxygen Sensing ◽  
Signal Transduction System ◽  
Two Component ◽  
Two Component Signal Transduction

Kinetic and mechanistic analyses of new classes of inhibitors of two-component signal transduction systems using a coupled assay containing HpkA–DrrA from Thermotoga maritima

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 885-896 ◽  
Author(s):  
J. Estelle Foster ◽  
Qin Sheng ◽  
Jonathan R. McClain ◽  
Mark Bures ◽  
Thalia I. Nicas ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Histidine Kinase ◽  
Competitive Inhibition ◽  
Thermotoga Maritima ◽  
Phosphorylation Site ◽  
Phosphoryl Group ◽  
Two Component ◽  
Two Component Signal Transduction ◽  
Coupled Assay ◽  
Signal Transduction Systems

Two-component signal transduction systems (TCSs) play fundamental roles in bacterial survival and pathogenesis and have been proposed as targets for the development of novel classes of antibiotics. A new coupled assay was developed and applied to analyse the kinetic mechanisms of three new kinds of inhibitors of TCS function. The assay exploits the biochemical properties of the cognate HpkA–DrrA histidine kinase–response regulator pair from Thermotoga maritima and allows multiple turnovers of HpkA, linear formation of phosphorylated DrrA, and Michaelis–Menten analysis of inhibitors. The assay was validated in several ways, including confirmation of competitive inhibition by adenosine 5′-β,γ-imidotriphosphate (AMP-PNP). The coupled assay, autophosphorylation and chemical cross-linking were used to determine the mechanisms by which several compounds inhibit TCS function. A cyanoacetoacetamide showed non-competitive inhibition with respect to ATP concentration in the coupled assay. The cyanoacetoacetamide also inhibited autophosphorylation of histidine kinases from other bacteria, indicating that the coupled assay could detect general inhibitors of histidine kinase function. Inhibition of HpkA autophosphorylation by this compound was probably caused by aggregation of HpkA, consistent with a previous model for other hydrophobic compounds. In contrast, ethodin was a potent inhibitor of the combined assay, did not inhibit HpkA autophosphorylation, but still led to aggregation of HpkA. These data suggest that ethodin bound to the HpkA kinase and inhibited transfer of the phosphoryl group to DrrA. A peptide corresponding to the phosphorylation site of DrrA appeared to inhibit TCS function by a mechanism similar to that of ethodin, except that autophosphorylation was inhibited at high peptide concentrations. The latter mechanism of inhibition of TCS function is unusual and its analysis demonstrates the utility of these approaches to the kinetic analyses of additional new classes of inhibitors of TCS function.

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