The Pseudomonas aeruginosa DNR transcription factor: light and shade of nitric oxide-sensing mechanisms

2011 ◽  
Vol 39 (1) ◽  
pp. 294-298 ◽  
Author(s):  
Giorgio Giardina ◽  
Nicoletta Castiglione ◽  
Manuela Caruso ◽  
Francesca Cutruzzolà ◽  
Serena Rinaldo
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Regulatory Protein ◽  
Structural Data ◽  
Activation Mechanism ◽  
Receptor Protein ◽  
Nitrate Respiration ◽  
Global Changes ◽  
Low Oxygen ◽  
Haem Protein

In response to environmental conditions, NO (nitric oxide) induces global changes in the cellular metabolism of Pseudomonas aeruginosa, which are strictly related to pathogenesis. In particular, at low oxygen tensions and in the presence of NO the denitrification alternative respiration is activated by a key regulator: DNR (dissimilative nitrate respiration regulator). DNR belongs to the CRP (cAMP receptor protein)–FNR (fumarate and nitrate reductase regulatory protein) superfamily of bacterial transcription factors. These regulators are involved in many different pathways and distinct activation mechanism seems to be operative in several cases. Recent results indicate that DNR is a haem protein capable of discriminating between NO and CO (carbon monoxide). On the basis of the available structural data, a suggested activation mechanism is discussed.

scholarly journals Amino Acid-Mediated Induction of the Basic Amino Acid-Specific Outer Membrane Porin OprD from Pseudomonas aeruginosa

1999 ◽  
Vol 181 (17) ◽  
pp. 5426-5432 ◽  
Author(s):  
Martina M. Ochs ◽  
Chung-Dar Lu ◽  
Robert E. W. Hancock ◽  
Ahmed T. Abdelal
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Regulatory Protein ◽  
Basic Amino Acid ◽  
Sole Source ◽  
Activation Mechanism ◽  
Beta Lactam ◽  
Mobility Shift ◽  
Carbon And Nitrogen

ABSTRACT Pseudomonas aeruginosa can utilize arginine and other amino acids as both carbon and nitrogen sources. Earlier studies have shown that the specific porin OprD facilitates the diffusion of basic amino acids as well as the structurally analogous beta-lactam antibiotic imipenem. The studies reported here showed that the expression of OprD was strongly induced when arginine, histidine, glutamate, or alanine served as the sole source of carbon. The addition of succinate exerted a negative effect on induction ofoprD, likely due to catabolite repression. The arginine-mediated induction was dependent on the regulatory protein ArgR, and binding of purified ArgR to its operator upstream of theoprD gene was demonstrated by gel mobility shift and DNase assays. The expression of OprD induced by glutamate as the carbon source, however, was independent of ArgR, indicating the presence of more than a single activation mechanism. In addition, it was observed that the levels of OprD responded strongly to glutamate and alanine as the sole sources of nitrogen. Thus, that the expression ofoprD is linked to both carbon and nitrogen metabolism ofPseudomonas aeruginosa.

scholarly journals Structure of heme d 1-free cd 1 nitrite reductase NirS

2020 ◽  
Vol 76 (6) ◽  
pp. 250-256
Author(s):  
Thomas Klünemann ◽  
Wulf Blankenfeldt
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Nitrite Reductase ◽  
Active Site ◽  
Opportunistic Pathogen ◽  
Active Enzyme ◽  
Free Form ◽  
Nitrate Respiration ◽  
Gram Negative ◽  
Open Conformation

A key step in anaerobic nitrate respiration is the reduction of nitrite to nitric oxide, which is catalysed by the cd 1 nitrite reductase NirS in, for example, the Gram-negative opportunistic pathogen Pseudomonas aeruginosa. Each subunit of this homodimeric enzyme consists of a cytochrome c domain and an eight-bladed β-propeller that binds the uncommon isobacteriochlorin heme d 1 as an essential part of its active site. Although NirS has been well studied mechanistically and structurally, the focus of previous studies has been on the active heme d 1-bound form. The heme d 1-free form of NirS reported here, which represents a premature state of the reductase, adopts an open conformation with the cytochrome c domains moved away from each other with respect to the active enzyme. Further, the movement of a loop around Trp498 seems to be related to a widening of the propeller, allowing easier access to the heme d 1-binding side. Finally, a possible link between the open conformation of NirS and flagella formation in P. aeruginosa is discussed.

A multitude of CRP/FNR-like transcription proteins in Bradyrhizobium japonicum

2006 ◽  
Vol 34 (1) ◽  
pp. 156-159 ◽  
Author(s):  
S. Mesa ◽  
H. Hennecke ◽  
H.-M. Fischer
Keyword(s):  
Gene Expression ◽  
Bradyrhizobium Japonicum ◽  
Sequence Similarity ◽  
Regulatory Protein ◽  
Regulatory System ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Low Oxygen ◽  
Denitrification Gene ◽  
Camp Receptor

In Bradyrhizobium japonicum, the nitrogen-fixing soya bean endosymbiont and facultative denitrifier, three CRP (cAMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein)-type transcription factors [FixK1, FixK2 and NnrR (nitrite and nitric oxide reductase regulator)] have been studied previously in the context of the regulation of nitrogen fixation and denitrification. The gene expression of both fixK1 and nnrR depends on FixK2, which acts as a key distributor of the ‘low-oxygen’ signal perceived by the two-component regulatory system FixLJ. While the targets for FixK1 are not known, NnrR transduces the nitrogen oxide signal to the level of denitrification gene expression. Besides these three regulators, the complete genome sequence of this organism has revealed the existence of 13 additional CRP/FNR-type proteins whose functions have not yet been studied. Based on sequence similarity and phylogenetic analysis, we discuss in this paper the peculiarities of these additional factors.

N-oxide sensing in Pseudomonas aeruginosa: expression and preliminary characterization of DNR, an FNR–CRP type transcriptional regulator

2005 ◽  
Vol 33 (1) ◽  
pp. 184-186 ◽  
Author(s):  
S. Rinaldo ◽  
G. Giardina ◽  
M. Brunori ◽  
F. Cutruzzolà
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcriptional Regulator ◽  
Receptor Protein ◽  
Nitrate Respiration ◽  
Camp Receptor Protein ◽  
Tight Control ◽  
Aggregation State ◽  
Hydrophobic Compounds ◽  
Camp Receptor

In denitrifying bacteria, the concentration of NO is maintained low by a tight control of the expression and activity of nitrite and NO reductases. Regulation involves redox-linked transcription factors, such as those belonging to the CRP-FNR (cAMP receptor protein–fumarate and nitrate reductase regulator) superfamily, which act as oxygen and N-oxide sensors. Given that few members of this superfamily have been characterized in detail, we have cloned, expressed and purified the dissimilative nitrate respiration regulator from Pseudomonas aeruginosa. To gain insights on the structural properties of the dissimilative nitrate respiration regulator, we have also determined the aggregation state of the purified protein and its ability to bind hydrophobic compounds such as 8-anilino-1-naphthalenesulphonic acid.

scholarly journals Modulation of Pseudomonas aeruginosa Biofilm Dispersal by a Cyclic-Di-GMP Phosphodiesterase with a Putative Hypoxia-Sensing Domain

2010 ◽  
Vol 76 (24) ◽  
pp. 8160-8173 ◽  
Author(s):  
Shuwen An ◽  
Ji'en Wu ◽  
Lian-Hui Zhang
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Regulatory Protein ◽  
Phosphodiesterase Activity ◽  
Low Oxygen ◽  
Ggdef Domain ◽  
Biofilm Dispersal ◽  
Biofilm Development

ABSTRACT Pseudomonas aeruginosa encodes many enzymes that are potentially associated with the synthesis or degradation of the widely conserved second messenger cyclic-di-GMP (c-di-GMP). In this study, we show that mutation of rbdA, which encodes a fusion protein consisting of PAS-PAC-GGDEF-EAL multidomains, results in decreased biofilm dispersal. RbdA contains a highly conserved GGDEF domain and EAL domain, which are involved in the synthesis and degradation of c-di-GMP, respectively. However, in vivo and in vitro analyses show that the full-length RbdA protein only displays phosphodiesterase activity, causing c-di-GMP degradation. Further analysis reveals that the GGDEF domain of RbdA plays a role in activating the phosphodiesterase activity of the EAL domain in the presence of GTP. Moreover, we show that deletion of the PAS domain or substitution of the key residues implicated in sensing low-oxygen stress abrogates the functionality of RbdA. Subsequent study showed that RbdA is involved in positive regulation of bacterial motility and production of rhamnolipids, which are associated with biofilm dispersal, and in negative regulation of production of exopolysaccharides, which are required for biofilm formation. These data indicate that the c-di-GMP-degrading regulatory protein RbdA promotes biofilm dispersal through its two-pronged effects on biofilm development, i.e., downregulating biofilm formation and upregulating production of the factors associated with biofilm dispersal.

scholarly journals The ArgR Regulatory Protein, a Helper to the Anaerobic Regulator ANR during Transcriptional Activation of thearcD Promoter in Pseudomonas aeruginosa

1999 ◽  
Vol 181 (8) ◽  
pp. 2459-2464 ◽  
Author(s):  
Chung-Dar Lu ◽  
Harald Winteler ◽  
Ahmed Abdelal ◽  
Dieter Haas
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Regulatory Protein ◽  
Protein A ◽  
Arginine Deiminase ◽  
Physical Contact ◽  
Sequence Motif ◽  
Low Oxygen ◽  
Mobility Shift

ABSTRACT Pseudomonas aeruginosa, when deprived of oxygen, generates ATP from arginine catabolism by enzymes of the arginine deiminase pathway, encoded by the arcDABC operon. Under conditions of low oxygen tension, the transcriptional activator ANR binds to a site centered 41.5 bp upstream of the arcDtranscriptional start. ANR-mediated anaerobic induction was enhanced two- to threefold by extracellular arginine. This arginine effect depended, in trans, on the transcriptional regulator ArgR and, in cis, on an ArgR binding site centered at −73.5 bp in the arcD promoter. Binding of purified ArgR protein to this site was demonstrated by electrophoretic mobility shift assays and DNase I footprinting. This ArgR recognition site contained a sequence, 5′-TGACGC-3′, which deviated in only 1 base from the common sequence motif 5′-TGTCGC-3′ found in other ArgR binding sites of P. aeruginosa. Furthermore, an alignment of all known ArgR binding sites confirmed that they consist of two directly repeated half-sites. In the absence of ANR, arginine did not induce thearc operon, suggesting that ArgR alone does not activate the arcD promoter. According to a model proposed, ArgR makes physical contact with ANR and thereby facilitates initiation ofarc transcription.

scholarly journals A Nitric Oxide-Responsive Transcriptional Regulator NsrR Cooperates With Lrp and CRP to Tightly Control the hmpA Gene in Vibrio vulnificus

2021 ◽  
Vol 12 ◽  
Author(s):  
Garam Choi ◽  
Dukyun Kim ◽  
Hanhyeok Im ◽  
Sang Ho Choi
Keyword(s):  
Nitric Oxide ◽  
Stress Responses ◽  
Promoter Region ◽  
Nitrosative Stress ◽  
Vibrio Vulnificus ◽  
Regulatory Protein ◽  
Transcriptional Regulator ◽  
Receptor Protein ◽  
Dependent Manner ◽  
Successful Infection

Nitric oxide (NO) is an important antimicrobial effector produced by the host innate immune system to counteract invading pathogens. To survive and establish a successful infection, a fulminating human pathogen Vibrio vulnificus expresses the hmpA gene encoding an NO dioxygenase in an NO-responsive manner. In this study, we identified an Rrf2-family transcriptional regulator NsrR that is predicted to contain the Fe-S cluster coordinated by three cysteine residues. Transcriptome analysis showed that NsrR controls the expression of multiple genes potentially involved in nitrosative stress responses. Particularly, NsrR acts as a strong repressor of hmpA transcription and relieves the repression of hmpA upon exposure to NO. Notably, nsrR and hmpA are transcribed divergently, and their promoter regions overlap with each other. Molecular biological analyses revealed that NsrR directly binds to this overlapping promoter region, which is alleviated by loss of the Fe-S cluster, leading to the subsequent derepression of hmpA under nitrosative stress. We further found that a leucine-responsive regulatory protein (Lrp) negatively regulates hmpA in an NsrR-dependent manner by directly binding to the promoter region, presumably resulting in a DNA conformation change to support the repression by NsrR. Meanwhile, a cyclic AMP receptor protein (CRP) positively regulates hmpA probably through repression of nsrR and lrp by directly binding to each promoter region in a sequential cascade. Altogether, this collaborative regulation of NsrR along with Lrp and CRP enables an elaborate control of hmpA transcription, contributing to survival under host-derived nitrosative stress and thereby the pathogenesis of V. vulnificus.

scholarly journals Transcriptional Regulation of the Flavohemoglobin Gene for Aerobic Nitric Oxide Detoxification by the Second Nitric Oxide-Responsive Regulator of Pseudomonas aeruginosa

2005 ◽  
Vol 187 (12) ◽  
pp. 3960-3968 ◽  
Author(s):  
Hiroyuki Arai ◽  
Michiko Hayashi ◽  
Azusa Kuroi ◽  
Masaharu Ishii ◽  
Yasuo Igarashi
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Mutant Strain ◽  
Regulatory Gene ◽  
Physiological Role ◽  
Nitrate Respiration ◽  
Binding Motif ◽  
Aerobic Conditions ◽  
Mutant Strains ◽  
Denitrification Genes

ABSTRACT The regulatory gene for a σ54-dependent-type transcriptional regulator, fhpR, is located upstream of the fhp gene for flavohemoglobin in Pseudomonas aeruginosa. Transcription of fhp was induced by nitrate, nitrite, nitric oxide (NO), and NO-generating reagents. Analysis of the fhp promoter activity in mutant strains deficient in the denitrification enzymes indicated that the promoter was regulated by NO or related reactive nitrogen species. The NO-responsive regulation was operative in a mutant strain deficient in DNR (dissimilatory nitrate respiration regulator), which is the NO-responsive regulator required for expression of the denitrification genes. A binding motif for σ54 was found in the promoter region of fhp, but an FNR (fumarate nitrate reductase regulator) box was not. The fhp promoter was inactive in the fhpR or rpoN mutant strain, suggesting that the NO-sensing regulation of the fhp promoter was mediated by FhpR. The DNR-dependent denitrification promoters (nirS, norC, and nosR) were active in the fhpR or rpoN mutants. These results indicated that P. aeruginosa has at least two independent NO-responsive regulatory systems. The fhp or fhpR mutant strains showed sensitivity to NO-generating reagents under aerobic conditions but not under anaerobic conditions. These mutants also showed significantly low aerobic NO consumption activity, indicating that the physiological role of flavohemoglobin in P. aeruginosa is detoxification of NO under aerobic conditions.

scholarly journals Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

2019 ◽  
Vol 75 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Odel Soren ◽  
Ardeshir Rineh ◽  
Diogo G Silva ◽  
Yuming Cai ◽  
Robert P Howlin ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Nitric Oxide Donor ◽  
Confocal Laser ◽  
Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

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