scholarly journals Tripartite Regulation of the glpFKD Operon Involved in Glycerol Catabolism by GylR, Crp, and SigF in Mycobacterium smegmatis

2019 ◽  
Vol 201 (24) ◽  
Author(s):  
Hyun-Ju Bong ◽  
Eon-Min Ko ◽  
Su-Yeon Song ◽  
In-Jeong Ko ◽  
Jeong-Il Oh
Keyword(s):  
Energy State ◽  
Transcriptional Activator ◽  
Glycerol Kinase ◽  
Receptor Protein ◽  
Upstream Region ◽  
Glycerol Metabolism ◽  
Dihydroxyacetone Phosphate ◽  
Content Type ◽  
Cellular Energy ◽  
Glycerol 3 Phosphate Dehydrogenase

ABSTRACT The glpD (MSMEG_6761) gene encoding glycerol-3-phosphate dehydrogenase was shown to be crucial for M. smegmatis to utilize glycerol as the sole carbon source. The glpD gene likely forms the glpFKD operon together with glpF and glpK, encoding a glycerol facilitator and glycerol kinase, respectively. The gylR (MSMEG_6757) gene, whose product belongs to the IclR family of transcriptional regulators, was identified 182 bp upstream of glpF. It was demonstrated that GylR serves as a transcriptional activator and is involved in the induction of glpFKD expression in the presence of glycerol. Three GylR-binding sites with the consensus sequence (GKTCGRC-N3-GYCGAMC) were identified in the upstream region of glpF by DNase I footprinting analysis. The presence of glycerol-3-phosphate was shown to decrease the binding affinity of GylR to the glpF upstream region with changes in the quaternary structure of GylR from tetramer to dimer. Besides GylR, cAMP receptor protein (Crp) and an alternative sigma factor, SigF, are also implicated in the regulation of glpFKD expression. Crp functions as a repressor, while SigF induces expression of glpFKD under energy-limiting conditions. In conclusion, we suggest here that the glpFKD operon is under the tripartite control of GylR, SigF, and Crp, which enables M. smegmatis to integrate the availability of glycerol, cellular energy state, and cellular levels of cAMP to exquisitely control expression of the glpFKD operon involved in glycerol metabolism. IMPORTANCE Using genetic approaches, we first revealed that glycerol is catabolized through the glycolytic pathway after conversion to dihydroxyacetone phosphate in two sequential reactions catalyzed by glycerol kinase (GlpK) and flavin adenine dinucleotide (FAD)-containing glycerol-3-phosphate dehydrogenase (GlpD) in M. smegmatis. Our study also revealed that in addition to the GylR transcriptional activator that mediates the induction of the glpFKD operon by glycerol, the operon is regulated by SigF and Crp, which reflect the cellular energy state and cAMP level, respectively.

scholarly journals Sycrp2 Is Essential for Twitching Motility in the CyanobacteriumSynechocystissp. Strain PCC 6803

2018 ◽  
Vol 200 (21) ◽  
Author(s):  
Wei-Yu Song ◽  
Sha-Sha Zang ◽  
Zheng-Ke Li ◽  
Guo-Zheng Dai ◽  
Ke Liu ◽  
...  
Keyword(s):  
Cell Motility ◽  
Receptor Protein ◽  
Upstream Region ◽  
Twitching Motility ◽  
Camp Receptor Protein ◽  
Mobility Shift ◽  
Content Type ◽  
Receptor Proteins ◽  
Camp Receptor ◽  
Pcc 6803

ABSTRACTTwo cAMP receptor proteins (CRPs), Sycrp1 (encoded bysll1371) and Sycrp2 (encoded bysll1924), exist in the cyanobacteriumSynechocystissp. strain PCC 6803. Previous studies have demonstrated that Sycrp1 has binding affinity for cAMP and is involved in motility by regulating the formation of pili. However, the function of Sycrp2 remains unknown. Here, we report thatsycrp2disruption results in the loss of motility ofSynechocystissp. PCC 6803, and that the phenotype can be recovered by reintroducing thesycrp2gene into the genome ofsycrp2-disrupted mutants. Electron microscopy showed that thesycrp2-disrupted mutant lost the pilus apparatus on the cell surface, resulting in a lack of cell motility. Furthermore, the transcript level of thepilA9-pilA11operon (essential for cell motility and regulated by the cAMP receptor protein Sycrp1) was markedly decreased insycrp2-disrupted mutants compared with the wild-type strain. Moreover, yeast two-hybrid analysis and a pulldown assay demonstrated that Sycrp2 interacted with Sycrp1 to form a heterodimer and that Sycrp1 and Sycrp2 interacted with themselves to form homodimers. Gel mobility shift assays revealed that Sycrp1 specifically binds to the upstream region ofpilA9. Together, these findings indicate that inSynechocystissp. PCC 6803, Sycrp2 regulates the formation of pili and cell motility by interacting with Sycrp1.IMPORTANCEcAMP receptor proteins (CRPs) are widely distributed in cyanobacteria and play important roles in regulating gene expression. Although many cyanobacterial species have two cAMP receptor-like proteins, the functional links between them are unknown. Here, we found that Sycrp2 in the cyanobacteriumSynechocystissp. strain PCC 6803 is essential for twitching motility and that it interacts with Sycrp1, a known cAMP receptor protein involved with twitching motility. Our findings indicate that the two cAMP receptor-like proteins in cyanobacteria do not have functional redundancy but rather work together.

scholarly journals A MARTX Toxin rtxA Gene Is Controlled by Host Environmental Signals through a CRP-Coordinated Regulatory Network in Vibrio vulnificus

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Zee-Won Lee ◽  
Seung-Ho Hwang ◽  
Garam Choi ◽  
Kyung Ku Jang ◽  
Tae Hee Lee ◽  
...  
Keyword(s):  
Regulatory Network ◽  
Vibrio Vulnificus ◽  
Regulatory Region ◽  
Receptor Protein ◽  
Upstream Region ◽  
Toxin Gene ◽  
Content Type ◽  
Environmental Signals ◽  
Exogenous Glucose ◽  
Successful Infection

ABSTRACT A multifunctional autoprocessing repeats-in-toxin (MARTX) toxin plays an essential role in the virulence of many pathogens, including a fulminating human pathogen Vibrio vulnificus. H-NS and HlyU repress and derepress expression of the MARTX toxin gene rtxA in V. vulnificus, respectively. However, little is known about other regulatory proteins and environmental signals involved in rtxA regulation. In this study, we found that a leucine-responsive regulatory protein (Lrp) activates rtxA by binding directly and specifically to the rtxA promoter, PrtxA. Phased hypersensitivity resulting from DNase I cleavage of the PrtxA regulatory region suggests that Lrp probably induces DNA bending in PrtxA. Lrp activates PrtxA independently of H-NS and HlyU, and leucine inhibits Lrp binding to PrtxA and reduces the Lrp-mediated activation. Furthermore, a cyclic AMP receptor protein (CRP) represses PrtxA, and exogenous glucose relieves the CRP-mediated repression. Biochemical and mutational analyses demonstrated that CRP binds directly and specifically to the upstream region of PrtxA, which presumably alters the DNA conformation in PrtxA and thus represses rtxA. Moreover, CRP represses expression of lrp and hlyU by binding directly to their upstream regions, forming coherent feed-forward loops with Lrp and HlyU. In conclusion, expression of rtxA is controlled by a regulatory network comprising CRP, Lrp, H-NS, and HlyU in response to changes in host environmental signals such as leucine and glucose. This collaborative regulation enables the elaborate expression of rtxA, thereby enhancing the fitness and pathogenesis of V. vulnificus during the course of infection. IMPORTANCE A MARTX toxin, RtxA, is an essential virulence factor of many pathogens, including Vibrio species. H-NS and HlyU repress and derepress, respectively, rtxA expression of a life-threatening pathogen, Vibrio vulnificus. We found that Lrp directly activates rtxA independently of H-NS and HlyU, and leucine inhibits the Lrp-mediated activation of rtxA. Furthermore, we demonstrated that CRP represses rtxA but derepresses in the presence of exogenous glucose. CRP represses rtxA not only directly by binding to upstream of rtxA but also indirectly by repressing lrp and hlyU. This is the first report of a regulatory network comprising CRP, Lrp, H-NS, and HlyU, which coordinates the rtxA expression in response to environmental signals such as leucine and glucose during infection. This elaborate regulatory network will enhance the fitness of V. vulnificus and contribute to its successful infection within the host.

scholarly journals LPL/AQP7/GPD2 promotes glycerol metabolism under hypoxia and prevents cardiac dysfunction during ischemia.

2020 ◽  
Author(s):  
Sohta Ishihama ◽  
Tatsuya Yoshida ◽  
Satoya Yoshida ◽  
Noriyuki Ouchi ◽  
Yu Mori ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Cardiac Dysfunction ◽  
Myocardial Infarct ◽  
Atp Synthesis ◽  
Glycerol Metabolism ◽  
Dihydroxyacetone Phosphate ◽  
Myocardial Infarct Size ◽  
Glucose And Lipid Metabolism ◽  
Acute Mi ◽  
Glycerol 3 Phosphate Dehydrogenase

Abstract Fatty acid constitutes a major energy substrate in the heart to fuel contraction under aerobic conditions. Ischemia downregulates fatty acid metabolism to adapt to the limited oxygen supply and makes glucose the preferred substrate. However, the mechanism of the myocardial metabolic shift during ischemia remains unknown. Here, we show that cardiomyocyte secretion of lipoprotein lipase (LPL), a principal enzyme that converts triglycerides to free fatty acids and glycerol, increased during myocardial infarction (MI). Cardiomyocyte-specific LPL deficiency enhanced cardiac dysfunction and apoptosis following MI. Deficiency of aquaporin 7 (AQP7), a glycerol channel in cardiomyocytes, increased the myocardial infarct size and apoptosis in response to ischemia. Ischemic conditions activated glycerol-3-phosphate dehydrogenase 2 (GPD2), which converts glycerol-3-phosphate into dihydroxyacetone phosphate to facilitate ATP synthesis from glycerol. Conversely, GPD2 deficiency exacerbated cardiac dysfunction after acute MI. Together, these results identify that LPL/AQP7/GPD2-mediated glycerol metabolism plays an important role to bridge glucose and lipid metabolism in MI and prevent myocardial ischemia-related damage.

scholarly journals Glycerol-Mediated Repression of Glucose Metabolism and Glycerol Kinase as the Sole Route of Glycerol Catabolism in the Haloarchaeon Haloferax volcanii

2009 ◽  
Vol 191 (13) ◽  
pp. 4307-4315 ◽  
Author(s):  
Katherine E. Sherwood ◽  
David José Cano ◽  
Julie A. Maupin-Furlow
Keyword(s):  
Glucose Metabolism ◽  
Glycerol Kinase ◽  
Activity Level ◽  
Haloferax Volcanii ◽  
Glycerol Metabolism ◽  
Pcr Analysis ◽  
Carbon Utilization ◽  
Gene Encoding ◽  
Upstream Gene ◽  
Glycerol 3 Phosphate Dehydrogenase

ABSTRACT Although glycerol is the primary carbon source available to halophilic heterotrophic communities, little is known regarding haloarchaeal glycerol metabolism. In this study, a gene encoding a glycerol kinase homolog (glpK; HVO_1541) was deleted from the genome of the haloarchaeon Haloferax volcanii by a markerless knockout strategy. The glpK mutant, KS4, readily grew on yeast extract-peptone complex medium and glucose minimal medium but was incapable of growth on glycerol. Glycerol kinase activity was dependent on the glpK gene and readily detected in cells grown on glucose and/or glycerol, with the activity level higher in medium supplemented with glycerol (with or without glucose) than in medium with glucose alone. An analysis of carbon utilization revealed that glycerol suppressed the metabolism of glucose in both the parent H26 and glpK mutant strains, with catabolite repression more pronounced in the glycerol kinase mutant. Transcripts specific for glpK and an upstream gene, gpdA, encoding a homolog of glycerol-3-phosphate dehydrogenase subunit A, were upregulated (8- and 74-fold, respectively) in the presence of glycerol and glucose compared to those in the presence of glucose alone. Furthermore, glpK was transcriptionally linked to the gpdC gene of the putative glycerol-3-phosphate dehydrogenase operon (gpdABC), based on the findings of reverse transcriptase PCR analysis. The results presented here provide genetic and biochemical evidence that glycerol metabolism proceeds through a glycerol kinase encoded by glpK and suggest that a glycerol-3-phosphate dehydrogenase encoded by the upstream gpdABC operon is also involved in this pathway. Furthermore, our findings reveal a unique example of glycerol-induced repression of glucose metabolism in H. volcanii.

scholarly journals Glycerol Metabolism Is Important for Cytotoxicity of Mycoplasma pneumoniae

2008 ◽  
Vol 191 (3) ◽  
pp. 747-753 ◽  
Author(s):  
Claudine Hames ◽  
Sven Halbedel ◽  
Michael Hoppert ◽  
Joachim Frey ◽  
Jörg Stülke
Keyword(s):  
Hydrogen Peroxide ◽  
Mycoplasma Pneumoniae ◽  
Carbon Sources ◽  
Constitutive Expression ◽  
Glycerol Kinase ◽  
Glycerol Metabolism ◽  
Dihydroxyacetone Phosphate ◽  
Facilitated Diffusion ◽  
Glycolytic Intermediate ◽  
Glycerol Facilitator

ABSTRACT Glycerol is one of the few carbon sources that can be utilized by Mycoplasma pneumoniae. Glycerol metabolism involves uptake by facilitated diffusion, phosphorylation, and the oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate, a glycolytic intermediate. We have analyzed the expression of the genes involved in glycerol metabolism and observed constitutive expression irrespective of the presence of glycerol or preferred carbon sources. Similarly, the enzymatic activity of glycerol kinase is not modulated by HPr-dependent phosphorylation. This lack of regulation is unique among the bacteria for which glycerol metabolism has been studied so far. Two types of enzymes catalyze the oxidation of glycerol 3-phosphate: oxidases and dehydrogenases. Here, we demonstrate that the enzyme encoded by the M. pneumoniae glpD gene is a glycerol 3-phosphate oxidase that forms hydrogen peroxide rather than NADH2. The formation of hydrogen peroxide by GlpD is crucial for cytotoxic effects of M. pneumoniae. A glpD mutant exhibited a significantly reduced formation of hydrogen peroxide and a severely reduced cytotoxicity. Attempts to isolate mutants affected in the genes of glycerol metabolism revealed that only the glpD gene, encoding the glycerol 3-phosphate oxidase, is dispensable. In contrast, the glpF and glpK genes, encoding the glycerol facilitator and the glycerol kinase, respectively, are essential in M. pneumoniae. Thus, the enzymes of glycerol metabolism are crucial for the pathogenicity of M. pneumoniae but also for other essential, yet-to-be-identified functions in the M. pneumoniae cell.

scholarly journals Mutations That StimulateflhDCExpression in Escherichia coli K-12

2015 ◽  
Vol 197 (19) ◽  
pp. 3087-3096 ◽  
Author(s):  
Karen A. Fahrner ◽  
Howard C. Berg
Keyword(s):  
Escherichia Coli ◽  
Transcription Factors ◽  
Insertion Sequence ◽  
Receptor Protein ◽  
Upstream Region ◽  
Single Nucleotide ◽  
Content Type ◽  
Sequence Elements ◽  
Camp Receptor ◽  
K 12

ABSTRACTMotility is a beneficial attribute that enables cells to access and explore new environments and to escape detrimental ones. The organelle of motility inEscherichia coliis the flagellum, and its production is initiated by the activating transcription factors FlhD and FlhC. The expression of these factors by theflhDCoperon is highly regulated and influenced by environmental conditions. TheflhDCpromoter is recognized by σ70and is dependent on the transcriptional activator cyclic AMP (cAMP)-cAMP receptor protein complex (cAMP-CRP). A number of K-12 strains exhibit limited motility due to low expression levels offlhDC. We report here a large number of mutations that stimulateflhDCexpression in such strains. They include single nucleotide changes in the −10 element of the promoter, in the promoter spacer, and in the cAMP-CRP binding region. In addition, we show that insertion sequence (IS) elements or a kanamycin gene located hundreds of base pairs upstream of the promoter can effectively enhance transcription, suggesting that the topology of a large upstream region plays a significant role in the regulation offlhDCexpression. None of the mutations eliminated the requirement for cAMP-CRP for activation. However, several mutations allowed expression in the absence of the nucleoid organizing protein, H-NS, which is normally required forflhDCexpression.IMPORTANCETheflhDCoperon ofEscherichia coliencodes transcription factors that initiate flagellar synthesis, an energetically costly process that is highly regulated. Few deregulating mutations have been reported thus far. This paper describes new single nucleotide mutations that stimulateflhDCexpression, including a number that map to the promoter spacer region. In addition, this work shows that insertion sequence elements or a kanamycin gene located far upstream from the promoter or repressor binding sites also stimulate transcription, indicating a role of regional topology in the regulation offlhDCexpression.

scholarly journals Glycerol metabolism in the neonatal rat

1970 ◽  
Vol 118 (3) ◽  
pp. 531-536 ◽  
Author(s):  
R. G. Vernon ◽  
D. G. Walker
Keyword(s):  
Kinase Activity ◽  
Neonatal Period ◽  
Glycerol Kinase ◽  
Neonatal Rat ◽  
Glycerol Metabolism ◽  
Kidney Slices ◽  
Period 2 ◽  
Liver And Kidney ◽  
Glycerol 3 Phosphate Dehydrogenase

1. The possible role of glycerol as a precursor in neonatal gluconeogenesis in the rat was investigated by recording the activities of glycerol kinase and l-glycerol 3-phosphate dehydrogenase in the liver, kidney and other tissues around birth and during the neonatal period. 2. Blood glycerol concentrations in the neonatal rat are high. 3. There is a marked increase after birth in the ability of both liver and kidney slices to convert glycerol into glucose plus glycogen that correlates with the increase in glycerol kinase activity. 4. High hepatic and renal l-glycerol 3-phosphate dehydrogenase activities are also found in the neonatal period. 5. The marked capacity for neonatal gluconeogenesis from glycerol thus demonstrated and the role of glycerol kinase in its control are discussed.

scholarly journals Cyclic AMP Receptor Protein RegulatescspE, an Early Cold-Inducible Gene, in Escherichia coli

2011 ◽  
Vol 193 (22) ◽  
pp. 6142-6151 ◽  
Author(s):  
Sheetal Uppal ◽  
Svetlana R. Maurya ◽  
Ramesh S. Hire ◽  
Narendra Jawali
Keyword(s):  
Escherichia Coli ◽  
Low Temperature ◽  
Cyclic Amp ◽  
Target Site ◽  
Class I ◽  
Receptor Protein ◽  
Upstream Region ◽  
Core Motif ◽  
Content Type ◽  
Cyclic Amp Receptor Protein

cspE, a member of thecspAfamily of cold shock proteins inEscherichia coli, is an early cold-inducible protein. The nucleic acid melting ability and transcription antiterminator activity of CspE have been reported to be critical for growth at low temperature. Here, we show that the cyclic AMP receptor protein (CRP), a global regulator involved in sugar metabolism, upregulatescspEinE. coli. Sequence analysis of thecspEupstream region revealed a putative CRP target site centered at −61.5 relative to the transcription start. The binding of CRP to this target site was demonstrated using electrophoretic mobility shift assays. The presence of this site was shown to be essential for PcspEactivation by CRP. Mutational analysis of the binding site indicated that the presence of an intact second core motif is more important than the first core motif for CRP-PcspEinteraction. Based on the promoter architecture, we classified PcspEas a class I CRP-dependent promoter. This was further substantiated by our data demonstrating the involvement of the AR1 domain of CRP in PcspEtranscription. Furthermore, the substitutions in the key residues of the RNA polymerase α-subunit C-terminal domain (α-CTD), which are important for class I CRP-dependent transcription, showed the involvement of 265 and 287 determinants in PcspEtranscription. In addition, the deletion ofcrpled to a growth defect at low temperature, suggesting that CRP plays an important role in cold adaptation.

scholarly journals Cyclic AMP (cAMP) Receptor Protein-cAMP Complex Regulates Heparosan Production in Escherichia coli Strain Nissle 1917

2015 ◽  
Vol 81 (22) ◽  
pp. 7687-7696 ◽  
Author(s):  
Huihui Yan ◽  
Feifei Bao ◽  
Liping Zhao ◽  
Yanying Yu ◽  
Jiaqin Tang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Carbon Sources ◽  
Coli Strain ◽  
Site Directed Mutagenesis ◽  
Receptor Protein ◽  
Upstream Region ◽  
Camp Receptor Protein ◽  
Content Type ◽  
Camp Receptor

ABSTRACTHeparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The availability of heparosan is a significant concern for the cost-effective synthesis of bioengineered heparin. The carbon source is known as the pivotal factor affecting heparosan production. However, the mechanism by which carbon sources control the biosynthesis of heparosan is unclear. In this study, we found that the biosynthesis of heparosan was influenced by different carbon sources. Glucose inhibits the biosynthesis of heparosan, while the addition of either fructose or mannose increases the yield of heparosan. Further study demonstrated that the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex binds to the upstream region of the region 3 promoter and stimulates the transcription of the gene cluster for heparosan biosynthesis. Site-directed mutagenesis of the CRP binding site abolished its capability of binding CRP and eliminated the stimulative effect on transcription.1H nuclear magnetic resonance (NMR) analysis was further performed to determine theEscherichia colistrain Nissle 1917 (EcN) heparosan structure and quantify extracellular heparosan production. Our results add to the understanding of the regulation of heparosan biosynthesis and may contribute to the study of other exopolysaccharide-producing strains.

scholarly journals Spo0A Suppresses sin Locus Expression in Clostridioides difficile

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Babita Adhikari Dhungel ◽  
Revathi Govind
Keyword(s):  
Diarrheal Disease ◽  
Toxin Production ◽  
The United States ◽  
Upstream Region ◽  
Pcr Analysis ◽  
Bacterial Cells ◽  
Master Regulator ◽  
Content Type ◽  
Clostridioides Difficile

ABSTRACT Clostridioides difficile is the leading cause of nosocomial infection and is the causative agent of antibiotic-associated diarrhea. The severity of the disease is directly associated with toxin production, and spores are responsible for the transmission and persistence of the organism. Previously, we characterized sin locus regulators SinR and SinR′ (we renamed it SinI), where SinR is the regulator of toxin production and sporulation. The SinI regulator acts as its antagonist. In Bacillus subtilis, Spo0A, the master regulator of sporulation, controls SinR by regulating the expression of its antagonist, sinI. However, the role of Spo0A in the expression of sinR and sinI in C. difficile had not yet been reported. In this study, we tested spo0A mutants in three different C. difficile strains, R20291, UK1, and JIR8094, to understand the role of Spo0A in sin locus expression. Western blot analysis revealed that spo0A mutants had increased SinR levels. Quantitative reverse transcription-PCR (qRT-PCR) analysis of its expression further supported these data. By carrying out genetic and biochemical assays, we show that Spo0A can bind to the upstream region of this locus to regulates its expression. This study provides vital information that Spo0A regulates the sin locus, which controls critical pathogenic traits such as sporulation, toxin production, and motility in C. difficile. IMPORTANCE Clostridioides difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. During infection, C. difficile spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. In C. difficile, the sin locus is known to regulate both sporulation and toxin production. In this study, we show that Spo0A, the master regulator of sporulation, controls sin locus expression. Results from our study suggest that Spo0A directly regulates the expression of this locus by binding to its upstream DNA region. This observation adds new detail to the gene regulatory network that connects sporulation and toxin production in this pathogen.

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