The Experts below are selected from a list of 252 Experts worldwide ranked by ideXlab platform

Christopher T Nomura - One of the best experts on this subject based on the ideXlab platform.

  • blocking the alternative Sigma Factor RpoN reduces virulence of pseudomonas aeruginosa isolated from cystic fibrosis patients and increases antibiotic sensitivity in a laboratory strain
    bioRxiv, 2018
    Co-Authors: Megan G Lloyd, Christopher T Nomura, James L Vossler, Jennifer F Moffat
    Abstract:

    Multidrug-resistant organisms (MDROs) are increasing in the health care setting, and there are few antimicrobial agents available to treat infections caused by these bacteria. Pseudomonas aeruginosa is an opportunistic pathogen in burn patients and individuals with cystic fibrosis (CF), and a leading cause of nosocomial infections. P. aeruginosa is inherently resistant to many antibiotics and can develop or acquire resistance to others, limiting options for treatment. P. aeruginosa has virulence Factors that are regulated by Sigma Factors in response to the tissue microenvironment. The alternative Sigma Factor, RpoN (s 54 ), regulates many virulence genes and is linked to antibiotic resistance. Recently, we described a cis-acting peptide, RpoN*, which acts as a 9molecular roadblock9, binding RpoN consensus promoters at the -24 site and blocking transcription. RpoN* reduces virulence of P. aeruginosa laboratory strains both in vitro and in vivo , but its effects in clinical isolates was not known. We investigated the effects of RpoN* on phenotypically varied P. aeruginosa strains isolated from cystic fibrosis patients. RpoN* expression reduced motility, biofilm formation, and pathogenesis in a P. aeruginosa - C. elegans infection model. RpoN* expression increased susceptibility to several beta-lactam based antibiotics in the lab strain P. aeruginosa PA19660 Xen5 . Here, we show that using a cis-acting peptide to block RpoN consensus promoters has potential clinical implications in reducing virulence and enhancing the activity of antibiotics.

  • Targeting the alternative Sigma Factor RpoN to combat virulence in Pseudomonas aeruginosa
    Scientific Reports, 2017
    Co-Authors: Megan G Lloyd, Benjamin R Lundgren, Jennifer F Moffat, Clayton W. Hall, Luke B.-p. Gagnon, Christopher T Nomura
    Abstract:

    Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that infects immunocompromised and cystic fibrosis patients. Treatment is difficult due to antibiotic resistance, and new antimicrobials are needed to treat infections. The alternative Sigma Factor 54 (σ^54, RpoN), regulates many virulence-associated genes. Thus, we evaluated inhibition of virulence in P. aeruginosa by a designed peptide (RpoN molecular roadblock, RpoN*) which binds specifically to RpoN consensus promoters. We expected that RpoN* binding to its consensus promoter sites would repress gene expression and thus virulence by blocking RpoN and/or other transcription Factors. RpoN* reduced transcription of approximately 700 genes as determined by microarray analysis, including genes related to virulence. RpoN* expression significantly reduced motility, protease secretion, pyocyanin and pyoverdine production, rhamnolipid production, and biofilm formation. Given the effectiveness of RpoN* in vitro , we explored its effects in a Caenorhabditis elegans–P. aeruginosa infection model. Expression of RpoN* protected C. elegans in a paralytic killing assay, whereas worms succumbed to paralysis and death in its absence. In a slow killing assay, which mimics establishment and proliferation of an infection, C. elegans survival was prolonged when RpoN* was expressed. Thus, blocking RpoN consensus promoter sites is an effective strategy for abrogation of P. aeruginosa virulence.

  • DdaR (PA1196) Regulates Expression of Dimethylarginine Dimethylaminohydrolase for the Metabolism of Methylarginines in Pseudomonas aeruginosa PAO1.
    Journal of Bacteriology, 2017
    Co-Authors: Benjamin R Lundgren, Frank J. Bailey, Gabriella Moley, Christopher T Nomura
    Abstract:

    : Dimethylarginine dimethylaminohydrolases (DDAHs) catalyze the hydrolysis of methylarginines to yield l-citrulline and methylamines as products. DDAHs and their central roles in methylarginine metabolism have been characterized for eukaryotic cells. While DDAHs are known to exist in some bacteria, including Streptomyces coelicolor and Pseudomonas aeruginosa, the physiological importance and genetic regulation of bacterial DDAHs remain poorly understood. To provide some insight into bacterial methylarginine metabolism, this study focused on identifying the key elements or Factors regulating DDAH expression in P. aeruginosa PAO1. First, results revealed that P. aeruginosa can utilize NG ,NG -dimethyl-l-arginine (ADMA) as a sole source of nitrogen but not carbon. Second, expression of the ddaH gene was observed to be induced in the presence of methylarginines, including NG -monomethyl-l-arginine (l-NMMA) and ADMA. Third, induction of the ddaH gene was shown to be achieved through a mechanism consisting of the putative enhancer-binding protein PA1196 and the alternative Sigma Factor RpoN. Both PA1196 and RpoN were essential for the expression of the ddaH gene in response to methylarginines. On the basis of the results of this study, PA1196 was given the name DdaR, for dimethylarginine dimethylaminohydrolase regulator. Interestingly, DdaR and its target ddaH gene are conserved only among P. aeruginosa strains, suggesting that this particular Pseudomonas species has evolved to utilize methylarginines from its environment.IMPORTANCE Methylated arginine residues are common constituents of eukaryotic proteins. During proteolysis, methylarginines are released in their free forms and become accessible nutrients for bacteria to utilize as growth substrates. In order to have a clearer and better understanding of this process, we explored methylarginine utilization in the metabolically versatile bacterium Pseudomonas aeruginosa PAO1. Our results show that the transcriptional regulator DdaR (PA1196) and the Sigma Factor RpoN positively regulate expression of dimethylarginine dimethylaminohydrolases (DDAHs) in response to exogenous methylarginines. DDAH is the central enzyme of methylarginine degradation, and its transcriptional regulation by DdaR-RpoN is expected to be conserved among P. aeruginosa strains.

  • ethanolamine catabolism in pseudomonas aeruginosa pao1 is regulated by the enhancer binding protein eatr pa4021 and the alternative Sigma Factor RpoN
    Journal of Bacteriology, 2016
    Co-Authors: Benjamin R Lundgren, Zaara Sarwar, Atahualpa Pinto, Jack G Ganley, Christopher T Nomura
    Abstract:

    ABSTRACT Although genes encoding enzymes and proteins related to ethanolamine catabolism are widely distributed in the genomes of Pseudomonas spp., ethanolamine catabolism has received little attention among this metabolically versatile group of bacteria. In an attempt to shed light on this subject, this study focused on defining the key regulatory Factors that govern the expression of the central ethanolamine catabolic pathway in Pseudomonas aeruginosa PAO1. This pathway is encoded by the PA4022-eat-eutBC operon and consists of a transport protein (Eat), an ethanolamine-ammonia lyase (EutBC), and an acetaldehyde dehydrogenase (PA4022). EutBC is an essential enzyme in ethanolamine catabolism because it hydrolyzes this amino alcohol into ammonia and acetaldehyde. The acetaldehyde intermediate is then converted into acetate in a reaction catalyzed by acetaldehyde dehydrogenase. Using a combination of growth analyses and β-galactosidase fusions, the enhancer-binding protein PA4021 and the Sigma Factor RpoN were shown to be positive regulators of the PA4022-eat-eutBC operon in P. aeruginosa PAO1. PA4021 and RpoN were required for growth on ethanolamine, and both of these regulatory proteins were essential for induction of the PA4022-eat-eutBC operon. Unexpectedly, the results indicate that acetaldehyde (and not ethanolamine) serves as the inducer molecule that is sensed by PA4021 and leads to the transcriptional activation of the PA4022-eat-eutBC operon. Due to its regulatory role in ethanolamine catabolism, PA4021 was given the name EatR. Both EatR and its target genes are conserved in several other Pseudomonas spp., suggesting that these bacteria share a mechanism for regulating ethanolamine catabolism. IMPORTANCE The results of this study provide a basis for understanding ethanolamine catabolism and its regulation in Pseudomonas aeruginosa PAO1. Interestingly, expression of the ethanolamine-catabolic genes in this bacterium was found to be under the control of a positive-feedback regulatory loop in a manner dependent on the transcriptional regulator PA4021, the Sigma Factor RpoN, and the metabolite acetaldehyde. Previously characterized regulators of ethanolamine catabolism are known to sense and respond directly to ethanolamine. In contrast, PA4021 (EatR) appears to monitor the intracellular levels of free acetaldehyde and responds through transcriptional activation of the ethanolamine-catabolic genes. This regulatory mechanism is unique and represents an alternative strategy used by bacteria to govern the acquisition of ethanolamine from their surroundings.

  • Defining the Metabolic Functions and Roles in Virulence of the RpoN1 and RpoN2 Genes in Ralstonia solanacearum GMI1000
    PLOS ONE, 2015
    Co-Authors: Benjamin R Lundgren, Morgan P. Connolly, Pratibha Choudhary, Tiffany S. Brookins-little, Snigdha Chatterjee, Ramesh Raina, Christopher T Nomura
    Abstract:

    The alternative Sigma Factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal RpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual RpoN genes. To determine if the RpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔRpoN1, ΔRpoN2 and ΔRpoN1 ΔRpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the RpoN1 gene. Furthermore, the RpoN1 gene was required for wilt disease on tomato whereas RpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of RpoN2 did not fully rescue the metabolic deficiencies of the ΔRpoN1 mutants; full recovery was specific to RpoN1. In comparison, only RpoN2 was able to genetically complement a ΔRpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000.

Megan G Lloyd - One of the best experts on this subject based on the ideXlab platform.

  • Blocking RpoN reduces virulence of Pseudomonas aeruginosa isolated from cystic fibrosis patients and increases antibiotic sensitivity in a laboratory strain
    Scientific Reports, 2019
    Co-Authors: Megan G Lloyd, James L Vossler, C. T. Nomura, Jennifer F Moffat
    Abstract:

    Multidrug-resistant organisms are increasing in healthcare settings, and there are few antimicrobials available to treat infections from these bacteria . Pseudomonas aeruginosa is an opportunistic pathogen in burn patients and individuals with cystic fibrosis (CF), and a leading cause of nosocomial infections. P. aeruginosa is inherently resistant to many antibiotics and can develop resistance to others, limiting treatment options. P. aeruginosa has multiple Sigma Factors to regulate transcription. The alternative Sigma Factor, RpoN (σ^54), regulates many virulence genes and is linked to antibiotic resistance. Recently, we described a cis-acting peptide, RpoN*, which is a “molecular roadblock”, binding consensus promoters at the -24 site, blocking transcription. RpoN* reduces virulence of P. aeruginosa laboratory strains, but its effects in clinical isolates was unknown. We investigated the effects of RpoN* on phenotypically varied P. aeruginosa strains isolated from CF patients. RpoN* expression reduced motility, biofilm formation, and pathogenesis in a P. aeruginosa-C. elegans infection model. Furthermore, we investigated RpoN* effects on antibiotic susceptibility in a laboratory strain. RpoN* expression increased susceptibility to several beta-lactam-based antibiotics in strain P. aeruginosa PA19660 Xen5 . We show that using a cis-acting peptide to block RpoN consensus promoters has potential clinical implications in reducing virulence and improving antibiotic susceptibility.

  • blocking the alternative Sigma Factor RpoN reduces virulence of pseudomonas aeruginosa isolated from cystic fibrosis patients and increases antibiotic sensitivity in a laboratory strain
    bioRxiv, 2018
    Co-Authors: Megan G Lloyd, Christopher T Nomura, James L Vossler, Jennifer F Moffat
    Abstract:

    Multidrug-resistant organisms (MDROs) are increasing in the health care setting, and there are few antimicrobial agents available to treat infections caused by these bacteria. Pseudomonas aeruginosa is an opportunistic pathogen in burn patients and individuals with cystic fibrosis (CF), and a leading cause of nosocomial infections. P. aeruginosa is inherently resistant to many antibiotics and can develop or acquire resistance to others, limiting options for treatment. P. aeruginosa has virulence Factors that are regulated by Sigma Factors in response to the tissue microenvironment. The alternative Sigma Factor, RpoN (s 54 ), regulates many virulence genes and is linked to antibiotic resistance. Recently, we described a cis-acting peptide, RpoN*, which acts as a 9molecular roadblock9, binding RpoN consensus promoters at the -24 site and blocking transcription. RpoN* reduces virulence of P. aeruginosa laboratory strains both in vitro and in vivo , but its effects in clinical isolates was not known. We investigated the effects of RpoN* on phenotypically varied P. aeruginosa strains isolated from cystic fibrosis patients. RpoN* expression reduced motility, biofilm formation, and pathogenesis in a P. aeruginosa - C. elegans infection model. RpoN* expression increased susceptibility to several beta-lactam based antibiotics in the lab strain P. aeruginosa PA19660 Xen5 . Here, we show that using a cis-acting peptide to block RpoN consensus promoters has potential clinical implications in reducing virulence and enhancing the activity of antibiotics.

  • Targeting the alternative Sigma Factor RpoN to combat virulence in Pseudomonas aeruginosa
    Scientific Reports, 2017
    Co-Authors: Megan G Lloyd, Benjamin R Lundgren, Jennifer F Moffat, Clayton W. Hall, Luke B.-p. Gagnon, Christopher T Nomura
    Abstract:

    Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that infects immunocompromised and cystic fibrosis patients. Treatment is difficult due to antibiotic resistance, and new antimicrobials are needed to treat infections. The alternative Sigma Factor 54 (σ^54, RpoN), regulates many virulence-associated genes. Thus, we evaluated inhibition of virulence in P. aeruginosa by a designed peptide (RpoN molecular roadblock, RpoN*) which binds specifically to RpoN consensus promoters. We expected that RpoN* binding to its consensus promoter sites would repress gene expression and thus virulence by blocking RpoN and/or other transcription Factors. RpoN* reduced transcription of approximately 700 genes as determined by microarray analysis, including genes related to virulence. RpoN* expression significantly reduced motility, protease secretion, pyocyanin and pyoverdine production, rhamnolipid production, and biofilm formation. Given the effectiveness of RpoN* in vitro , we explored its effects in a Caenorhabditis elegans–P. aeruginosa infection model. Expression of RpoN* protected C. elegans in a paralytic killing assay, whereas worms succumbed to paralysis and death in its absence. In a slow killing assay, which mimics establishment and proliferation of an infection, C. elegans survival was prolonged when RpoN* was expressed. Thus, blocking RpoN consensus promoter sites is an effective strategy for abrogation of P. aeruginosa virulence.

Heath F Damron - One of the best experts on this subject based on the ideXlab platform.

  • analysis of the pseudomonas aeruginosa regulon controlled by the sensor kinase kinb and Sigma Factor RpoN
    Journal of Bacteriology, 2012
    Co-Authors: Heath F Damron, Joshua P Owings, Yuta Okkotsu, John J Varga, Jill R Schurr, Joanna B Goldberg, Michael J Schurr, Hongwei D Yu
    Abstract:

    Alginate overproduction by Pseudomonas aeruginosa, also known as mucoidy, is associated with chronic endobronchial infections in cystic fibrosis. Alginate biosynthesis is initiated by the extracytoplasmic function Sigma Factor (σ22; AlgU/AlgT). In the wild-type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered to the cytoplasmic membrane by the anti-Sigma Factor MucA that inhibits alginate production. One mechanism underlying the conversion to mucoidy is mutation of mucA. However, the mucoid conversion can occur in wt mucA strains via the degradation of MucA by activated intramembrane proteases AlgW and/or MucP. Previously, we reported that the deletion of the sensor kinase KinB in PAO1 induces an AlgW-dependent proteolysis of MucA, resulting in alginate overproduction. This type of mucoid induction requires the alternate Sigma Factor RpoN (σ54). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB RpoN double mutant. In the kinB mutant of PAO1, RpoN controlled the expression of approximately 20% of the genome. In addition to alginate biosynthetic and regulatory genes, KinB and RpoN also control a large number of genes including those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, BALB/c mice exhibited increased survival when challenged with the kinB mutant relative to survival with PAO1 challenge. Together, these data strongly suggest that KinB regulates virulence Factors important for the development of acute pneumonia and conversion to mucoidy.

Jennifer F Moffat - One of the best experts on this subject based on the ideXlab platform.

  • Blocking RpoN reduces virulence of Pseudomonas aeruginosa isolated from cystic fibrosis patients and increases antibiotic sensitivity in a laboratory strain
    Scientific Reports, 2019
    Co-Authors: Megan G Lloyd, James L Vossler, C. T. Nomura, Jennifer F Moffat
    Abstract:

    Multidrug-resistant organisms are increasing in healthcare settings, and there are few antimicrobials available to treat infections from these bacteria . Pseudomonas aeruginosa is an opportunistic pathogen in burn patients and individuals with cystic fibrosis (CF), and a leading cause of nosocomial infections. P. aeruginosa is inherently resistant to many antibiotics and can develop resistance to others, limiting treatment options. P. aeruginosa has multiple Sigma Factors to regulate transcription. The alternative Sigma Factor, RpoN (σ^54), regulates many virulence genes and is linked to antibiotic resistance. Recently, we described a cis-acting peptide, RpoN*, which is a “molecular roadblock”, binding consensus promoters at the -24 site, blocking transcription. RpoN* reduces virulence of P. aeruginosa laboratory strains, but its effects in clinical isolates was unknown. We investigated the effects of RpoN* on phenotypically varied P. aeruginosa strains isolated from CF patients. RpoN* expression reduced motility, biofilm formation, and pathogenesis in a P. aeruginosa-C. elegans infection model. Furthermore, we investigated RpoN* effects on antibiotic susceptibility in a laboratory strain. RpoN* expression increased susceptibility to several beta-lactam-based antibiotics in strain P. aeruginosa PA19660 Xen5 . We show that using a cis-acting peptide to block RpoN consensus promoters has potential clinical implications in reducing virulence and improving antibiotic susceptibility.

  • blocking the alternative Sigma Factor RpoN reduces virulence of pseudomonas aeruginosa isolated from cystic fibrosis patients and increases antibiotic sensitivity in a laboratory strain
    bioRxiv, 2018
    Co-Authors: Megan G Lloyd, Christopher T Nomura, James L Vossler, Jennifer F Moffat
    Abstract:

    Multidrug-resistant organisms (MDROs) are increasing in the health care setting, and there are few antimicrobial agents available to treat infections caused by these bacteria. Pseudomonas aeruginosa is an opportunistic pathogen in burn patients and individuals with cystic fibrosis (CF), and a leading cause of nosocomial infections. P. aeruginosa is inherently resistant to many antibiotics and can develop or acquire resistance to others, limiting options for treatment. P. aeruginosa has virulence Factors that are regulated by Sigma Factors in response to the tissue microenvironment. The alternative Sigma Factor, RpoN (s 54 ), regulates many virulence genes and is linked to antibiotic resistance. Recently, we described a cis-acting peptide, RpoN*, which acts as a 9molecular roadblock9, binding RpoN consensus promoters at the -24 site and blocking transcription. RpoN* reduces virulence of P. aeruginosa laboratory strains both in vitro and in vivo , but its effects in clinical isolates was not known. We investigated the effects of RpoN* on phenotypically varied P. aeruginosa strains isolated from cystic fibrosis patients. RpoN* expression reduced motility, biofilm formation, and pathogenesis in a P. aeruginosa - C. elegans infection model. RpoN* expression increased susceptibility to several beta-lactam based antibiotics in the lab strain P. aeruginosa PA19660 Xen5 . Here, we show that using a cis-acting peptide to block RpoN consensus promoters has potential clinical implications in reducing virulence and enhancing the activity of antibiotics.

  • Targeting the alternative Sigma Factor RpoN to combat virulence in Pseudomonas aeruginosa
    Scientific Reports, 2017
    Co-Authors: Megan G Lloyd, Benjamin R Lundgren, Jennifer F Moffat, Clayton W. Hall, Luke B.-p. Gagnon, Christopher T Nomura
    Abstract:

    Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that infects immunocompromised and cystic fibrosis patients. Treatment is difficult due to antibiotic resistance, and new antimicrobials are needed to treat infections. The alternative Sigma Factor 54 (σ^54, RpoN), regulates many virulence-associated genes. Thus, we evaluated inhibition of virulence in P. aeruginosa by a designed peptide (RpoN molecular roadblock, RpoN*) which binds specifically to RpoN consensus promoters. We expected that RpoN* binding to its consensus promoter sites would repress gene expression and thus virulence by blocking RpoN and/or other transcription Factors. RpoN* reduced transcription of approximately 700 genes as determined by microarray analysis, including genes related to virulence. RpoN* expression significantly reduced motility, protease secretion, pyocyanin and pyoverdine production, rhamnolipid production, and biofilm formation. Given the effectiveness of RpoN* in vitro , we explored its effects in a Caenorhabditis elegans–P. aeruginosa infection model. Expression of RpoN* protected C. elegans in a paralytic killing assay, whereas worms succumbed to paralysis and death in its absence. In a slow killing assay, which mimics establishment and proliferation of an infection, C. elegans survival was prolonged when RpoN* was expressed. Thus, blocking RpoN consensus promoter sites is an effective strategy for abrogation of P. aeruginosa virulence.

Benjamin R Lundgren - One of the best experts on this subject based on the ideXlab platform.

  • Targeting the alternative Sigma Factor RpoN to combat virulence in Pseudomonas aeruginosa
    Scientific Reports, 2017
    Co-Authors: Megan G Lloyd, Benjamin R Lundgren, Jennifer F Moffat, Clayton W. Hall, Luke B.-p. Gagnon, Christopher T Nomura
    Abstract:

    Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that infects immunocompromised and cystic fibrosis patients. Treatment is difficult due to antibiotic resistance, and new antimicrobials are needed to treat infections. The alternative Sigma Factor 54 (σ^54, RpoN), regulates many virulence-associated genes. Thus, we evaluated inhibition of virulence in P. aeruginosa by a designed peptide (RpoN molecular roadblock, RpoN*) which binds specifically to RpoN consensus promoters. We expected that RpoN* binding to its consensus promoter sites would repress gene expression and thus virulence by blocking RpoN and/or other transcription Factors. RpoN* reduced transcription of approximately 700 genes as determined by microarray analysis, including genes related to virulence. RpoN* expression significantly reduced motility, protease secretion, pyocyanin and pyoverdine production, rhamnolipid production, and biofilm formation. Given the effectiveness of RpoN* in vitro , we explored its effects in a Caenorhabditis elegans–P. aeruginosa infection model. Expression of RpoN* protected C. elegans in a paralytic killing assay, whereas worms succumbed to paralysis and death in its absence. In a slow killing assay, which mimics establishment and proliferation of an infection, C. elegans survival was prolonged when RpoN* was expressed. Thus, blocking RpoN consensus promoter sites is an effective strategy for abrogation of P. aeruginosa virulence.

  • DdaR (PA1196) Regulates Expression of Dimethylarginine Dimethylaminohydrolase for the Metabolism of Methylarginines in Pseudomonas aeruginosa PAO1.
    Journal of Bacteriology, 2017
    Co-Authors: Benjamin R Lundgren, Frank J. Bailey, Gabriella Moley, Christopher T Nomura
    Abstract:

    : Dimethylarginine dimethylaminohydrolases (DDAHs) catalyze the hydrolysis of methylarginines to yield l-citrulline and methylamines as products. DDAHs and their central roles in methylarginine metabolism have been characterized for eukaryotic cells. While DDAHs are known to exist in some bacteria, including Streptomyces coelicolor and Pseudomonas aeruginosa, the physiological importance and genetic regulation of bacterial DDAHs remain poorly understood. To provide some insight into bacterial methylarginine metabolism, this study focused on identifying the key elements or Factors regulating DDAH expression in P. aeruginosa PAO1. First, results revealed that P. aeruginosa can utilize NG ,NG -dimethyl-l-arginine (ADMA) as a sole source of nitrogen but not carbon. Second, expression of the ddaH gene was observed to be induced in the presence of methylarginines, including NG -monomethyl-l-arginine (l-NMMA) and ADMA. Third, induction of the ddaH gene was shown to be achieved through a mechanism consisting of the putative enhancer-binding protein PA1196 and the alternative Sigma Factor RpoN. Both PA1196 and RpoN were essential for the expression of the ddaH gene in response to methylarginines. On the basis of the results of this study, PA1196 was given the name DdaR, for dimethylarginine dimethylaminohydrolase regulator. Interestingly, DdaR and its target ddaH gene are conserved only among P. aeruginosa strains, suggesting that this particular Pseudomonas species has evolved to utilize methylarginines from its environment.IMPORTANCE Methylated arginine residues are common constituents of eukaryotic proteins. During proteolysis, methylarginines are released in their free forms and become accessible nutrients for bacteria to utilize as growth substrates. In order to have a clearer and better understanding of this process, we explored methylarginine utilization in the metabolically versatile bacterium Pseudomonas aeruginosa PAO1. Our results show that the transcriptional regulator DdaR (PA1196) and the Sigma Factor RpoN positively regulate expression of dimethylarginine dimethylaminohydrolases (DDAHs) in response to exogenous methylarginines. DDAH is the central enzyme of methylarginine degradation, and its transcriptional regulation by DdaR-RpoN is expected to be conserved among P. aeruginosa strains.

  • ethanolamine catabolism in pseudomonas aeruginosa pao1 is regulated by the enhancer binding protein eatr pa4021 and the alternative Sigma Factor RpoN
    Journal of Bacteriology, 2016
    Co-Authors: Benjamin R Lundgren, Zaara Sarwar, Atahualpa Pinto, Jack G Ganley, Christopher T Nomura
    Abstract:

    ABSTRACT Although genes encoding enzymes and proteins related to ethanolamine catabolism are widely distributed in the genomes of Pseudomonas spp., ethanolamine catabolism has received little attention among this metabolically versatile group of bacteria. In an attempt to shed light on this subject, this study focused on defining the key regulatory Factors that govern the expression of the central ethanolamine catabolic pathway in Pseudomonas aeruginosa PAO1. This pathway is encoded by the PA4022-eat-eutBC operon and consists of a transport protein (Eat), an ethanolamine-ammonia lyase (EutBC), and an acetaldehyde dehydrogenase (PA4022). EutBC is an essential enzyme in ethanolamine catabolism because it hydrolyzes this amino alcohol into ammonia and acetaldehyde. The acetaldehyde intermediate is then converted into acetate in a reaction catalyzed by acetaldehyde dehydrogenase. Using a combination of growth analyses and β-galactosidase fusions, the enhancer-binding protein PA4021 and the Sigma Factor RpoN were shown to be positive regulators of the PA4022-eat-eutBC operon in P. aeruginosa PAO1. PA4021 and RpoN were required for growth on ethanolamine, and both of these regulatory proteins were essential for induction of the PA4022-eat-eutBC operon. Unexpectedly, the results indicate that acetaldehyde (and not ethanolamine) serves as the inducer molecule that is sensed by PA4021 and leads to the transcriptional activation of the PA4022-eat-eutBC operon. Due to its regulatory role in ethanolamine catabolism, PA4021 was given the name EatR. Both EatR and its target genes are conserved in several other Pseudomonas spp., suggesting that these bacteria share a mechanism for regulating ethanolamine catabolism. IMPORTANCE The results of this study provide a basis for understanding ethanolamine catabolism and its regulation in Pseudomonas aeruginosa PAO1. Interestingly, expression of the ethanolamine-catabolic genes in this bacterium was found to be under the control of a positive-feedback regulatory loop in a manner dependent on the transcriptional regulator PA4021, the Sigma Factor RpoN, and the metabolite acetaldehyde. Previously characterized regulators of ethanolamine catabolism are known to sense and respond directly to ethanolamine. In contrast, PA4021 (EatR) appears to monitor the intracellular levels of free acetaldehyde and responds through transcriptional activation of the ethanolamine-catabolic genes. This regulatory mechanism is unique and represents an alternative strategy used by bacteria to govern the acquisition of ethanolamine from their surroundings.

  • Defining the Metabolic Functions and Roles in Virulence of the RpoN1 and RpoN2 Genes in Ralstonia solanacearum GMI1000
    PLOS ONE, 2015
    Co-Authors: Benjamin R Lundgren, Morgan P. Connolly, Pratibha Choudhary, Tiffany S. Brookins-little, Snigdha Chatterjee, Ramesh Raina, Christopher T Nomura
    Abstract:

    The alternative Sigma Factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal RpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual RpoN genes. To determine if the RpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔRpoN1, ΔRpoN2 and ΔRpoN1 ΔRpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the RpoN1 gene. Furthermore, the RpoN1 gene was required for wilt disease on tomato whereas RpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of RpoN2 did not fully rescue the metabolic deficiencies of the ΔRpoN1 mutants; full recovery was specific to RpoN1. In comparison, only RpoN2 was able to genetically complement a ΔRpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000.

  • the metabolism of r 3 hydroxybutyrate is regulated by the enhancer binding protein pa2005 and the alternative Sigma Factor RpoN in pseudomonas aeruginosa pao1
    Microbiology, 2015
    Co-Authors: Benjamin R Lundgren, Zaara Sarwar, Joshua R Harris, Ryan A Scheel, Christopher T Nomura
    Abstract:

    A variety of soil-dwelling bacteria produce polyhydroxybutyrate (PHB), which serves as a source of energy and carbon under nutrient deprivation. Bacteria belonging to the genus Pseudomonas do not generally produce PHB but are capable of using the PHB degradation product (R)-3-hydroxybutyrate [(R)-3-HB] as a growth substrate. Essential to this utilization is the NAD+-dependent dehydrogenase BdhA that converts (R)-3-HB into acetoacetate, a molecule that readily enters central metabolism. Apart from the numerous studies that had focused on the biochemical characterization of BdhA, there was nothing known about the assimilation of (R)-3-HB in Pseudomonas, including the genetic regulation of bdhA expression. This study aimed to define the regulatory Factors that govern or dictate the expression of the bdhA gene and (R)-3-HB assimilation in Pseudomonas aeruginosa PAO1. Importantly, expression of the bdhA gene was found to be specifically induced by (R)-3-HB in a manner dependent on the alternative Sigma Factor RpoN and the enhancer-binding protein PA2005.This mode of regulation was essential for the utilization of (R)-3-HB as a sole source of energy and carbon. However, non-induced levels of bdhA expression were sufficient for P. aeruginosa PAO1 to grow on ( ± )-1,3-butanediol, which is catabolized through an (R)-3-HB intermediate. Because this is, we believe, the first report of an enhancer-binding protein that responds to (R)-3-HB, PA2005 was named HbcR for (R)-3-hydroxybutyrate catabolism regulator.