scholarly journals Effects of Nitrogen Dioxide and Anoxia on Global Gene and Protein Expression in Long-Term Continuous Cultures of Nitrosomonas eutropha C91

2012 ◽  
Vol 78 (14) ◽  
pp. 4788-4794 ◽  
Author(s):  
Boran Kartal ◽  
Hans J. C. T. Wessels ◽  
Erwin van der Biezen ◽  
Kees-Jan Francoijs ◽  
Mike S. M. Jetten ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Expression ◽  
Wastewater Treatment Plants ◽  
Batch Cultivation ◽  
Culture Conditions ◽  
Anaerobic Growth ◽  
Nitric Oxide Reductase ◽  
Content Type ◽  
Anoxic Conditions ◽  
Gene And Protein Expression

ABSTRACTNitrosomonas eutrophais an ammonia-oxidizing betaproteobacterium found in environments with high ammonium levels, such as wastewater treatment plants. The effects of NO2on gene and protein expression under oxic and anoxic conditions were determined by maintainingN. eutrophastrain C91 in a chemostat fed with ammonium under oxic, oxic-plus-NO2, and anoxic-plus-NO2culture conditions. Cells remained viable but ceased growing under anoxia; hence, the chemostat was switched from continuous to batch cultivation to retain biomass. After several weeks under each condition, biomass was harvested for total mRNA and protein isolation. Exposure ofN. eutrophaC91 to NO2under either oxic or anoxic conditions led to a decrease in proteins involved in N and C assimilation and storage and an increase in proteins involved in energy conservation, including ammonia monooxygenase (AmoCAB). Exposure to anoxia plus NO2resulted in increased representation of proteins and transcripts reflective of an energy-deprived state. Several proteins implicated in N-oxide metabolism were expressed and remained unchanged throughout the experiment, except for NorCB nitric oxide reductase, which was not detected in the proteome. Rather, NorY nitric oxide reductase was expressed under oxic-plus-NO2and anoxic-plus-NO2conditions. The results indicate that exposure to NO2results in an energy-deprived state ofN. eutrophaC91 and that anaerobic growth could not be supported with NO2as an oxidant.

Gene and Protein Expression Profiles ofShewanella oneidensisduring Anaerobic Growth with Different Electron Acceptors

2002 ◽  
Vol 6 (1) ◽  
pp. 39-60 ◽  
Author(s):  
Alex S. Beliaev ◽  
Dorothea K. Thompson ◽  
Tripti Khare ◽  
Hanjo Lim ◽  
Craig C. Brandt ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression Profiles ◽  
Electron Acceptors ◽  
Anaerobic Growth ◽  
Gene And Protein Expression ◽  
Protein Expression Profiles

scholarly journals Environmental and Genetic Determinants of Biofilm Formation inParacoccus denitrificans

mSphere ◽  
2017 ◽  
Vol 2 (5) ◽  
Author(s):  
Santosh Kumar ◽  
Stephen Spiro
Keyword(s):  
Nitric Oxide ◽  
Biofilm Formation ◽  
Paracoccus Denitrificans ◽  
Binding Protein ◽  
Anaerobic Growth ◽  
Type I ◽  
Content Type ◽  
Attached Growth ◽  
Polystyrene Surface ◽  
Biofilm Development

ABSTRACTThe genome of the denitrifying bacteriumParacoccus denitrificanspredicts the expression of a small heme-containing nitric oxide (NO) binding protein, H-NOX. The genome organization and prior work in other bacteria suggest that H-NOX interacts with a diguanylate cyclase that cyclizes GTP to make cyclic di-GMP (cdGMP). Since cdGMP frequently regulates attached growth as a biofilm, we first established conditions for biofilm development byP. denitrificans. We found that adhesion to a polystyrene surface is strongly stimulated by the addition of 10 mM Ca2+to rich media. The genome encodes at least 11 repeats-in-toxin family proteins that are predicted to be secreted by the type I secretion system (TISS). We deleted the genes encoding the TISS and found that the mutant is almost completely deficient for attached growth. Adjacent to the TISS genes there is a potential open reading frame encoding a 2,211-residue protein with 891 Asp-Ala repeats. This protein is also predicted to bind calcium and to be a TISS substrate, and a mutant specifically lacking this protein is deficient in biofilm formation. By analysis of mutants and promoter reporter fusions, we show that biofilm formation is stimulated by NO generated endogenously by the respiratory reduction of nitrite. A mutant lacking both predicted diguanylate cyclases encoded in the genome overproduces biofilm, implying that cdGMP is a negative regulator of attached growth. Our data are consistent with a model in which there are H-NOX-dependent and -independent pathways by which NO stimulates biofilm formation.IMPORTANCEThe bacteriumParacoccus denitrificansis a model for the process of denitrification, by which nitrate is reduced to dinitrogen during anaerobic growth. Denitrification is important for soil fertility and greenhouse gas emission and in waste and water treatment processes. The ability of bacteria to grow as a biofilm attached to a solid surface is important in many different contexts. In this paper, we report that attached growth ofP. denitrificansis stimulated by nitric oxide, an intermediate in the denitrification pathway. We also show that calcium ions stimulate attached growth, and we identify a large calcium binding protein that is required for growth on a polystyrene surface. We identify components of a signaling pathway through which nitric oxide may regulate biofilm formation. Our results point to an intimate link between metabolic processes and the ability ofP. denitrificansto grow attached to a surface.

scholarly journals Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses

2015 ◽  
Vol 82 (1) ◽  
pp. 51-61 ◽  
Author(s):  
Brian J. Vaccaro ◽  
Michael P. Thorgersen ◽  
W. Andrew Lancaster ◽  
Morgan N. Price ◽  
Kelly M. Wetmore ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitrous Oxide ◽  
Electron Acceptor ◽  
Pseudomonas Stutzeri ◽  
Basic Function ◽  
Nitric Oxide Reductase ◽  
Content Type ◽  
Nitric Oxide Release ◽  
Peripheral Proteins ◽  
Cofactor Biosynthesis

ABSTRACTEnzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library fromPseudomonas stutzeristrain RCH2 was grown under denitrifying conditions, using nitrate or nitrite as an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. The dominant HemN paralog involved in heme biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation.

Expression of Nitric Oxide Synthase Interacting Protein (NOSIP) is Decreased in the Pulmonary Vasculature of Nitrofen-Induced Congenital Diaphragmatic Hernia

2018 ◽  
Vol 29 (01) ◽  
pp. 102-107
Author(s):  
Hiroki Nakamura ◽  
Julia Zimmer ◽  
Florian Friedmacher ◽  
Prem Puri
Keyword(s):  
Nitric Oxide ◽  
Protein Expression ◽  
Congenital Diaphragmatic Hernia ◽  
Diaphragmatic Hernia ◽  
No Synthase ◽  
Laser Scanning Microscopy ◽  
Pulmonary Vasculature ◽  
No Production ◽  
Interacting Protein ◽  
Gene And Protein Expression

Introduction Persistent pulmonary hypertension (PPH) is a major cause of morbidity and mortality in newborns with congenital diaphragmatic hernia (CDH). PPH is characterized by increased vascular resistance and smooth muscle cell (SMC) proliferation, leading to obstructive changes in the pulmonary vasculature. Nitric oxide (NO), generated by endothelial NO synthase (eNOS), is an important regulator of vascular tone and plays a key role in pulmonary vasodilatation. NO synthase interacting protein (NOSIP), which is strongly expressed by pulmonary SMCs, has recently been identified to reduce the endogenous NO production by interacting with eNOS. We designed this study to investigate the pulmonary vascular expression of NOSIP in the nitrofen-induced CDH model. Materials and Methods Time-mated Sprague Dawley rats received nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and lung specimens divided into CDH and control (n = 6 for each group). Quantitative real-time polymerase chain reaction and Western blotting were performed to analyze pulmonary gene and protein expression of NOSIP. Immunofluorescence double staining for NOSIP was combined with a specific SMC marker to evaluate protein expression in the pulmonary vasculature. Results Relative messenger ribonucleic acid and protein expression of NOSIP was significantly decreased in nitrofen-exposed CDH lungs compared with controls. Confocal laser scanning microscopy revealed markedly diminished NOSIP immunofluorescence in nitrofen-exposed CDH lungs compared with controls, mainly in the muscular and endothelial components of the pulmonary vasculature. Conclusion This study demonstrates for the first time decreased NOSIP expression in the pulmonary vasculature of the nitrofen-induced CDH. These findings suggest that NOSIP underexpression may interfere with NO production, contributing to abnormal vascular remodeling and PPH.

scholarly journals Gonococcal Nitric Oxide Reductase Is Encoded by a Single Gene, norB, Which Is Required for Anaerobic Growth and Is Induced by Nitric Oxide

2000 ◽  
Vol 68 (9) ◽  
pp. 5241-5246 ◽  
Author(s):  
Tracey C. Householder ◽  
Elizabeth M. Fozo ◽  
Jean A. Cardinale ◽  
Virginia L. Clark
Keyword(s):  
Nitric Oxide ◽  
Ralstonia Eutropha ◽  
Single Gene ◽  
Nitrite Reduction ◽  
Anaerobic Growth ◽  
Nitric Oxide Donor ◽  
Nitric Oxide Reductase ◽  
Wild Type ◽  
Gene Encoding ◽  
Anaerobic Environment

ABSTRACT The gene encoding a nitric oxide reductase has been identified inNeisseria gonorrhoeae. The norB gene product shares significant identity with the nitric oxide reductases inRalstonia eutropha and Synechocystis sp. and, like those organisms, the gonococcus lacks a norC homolog. The gonococcal norB gene was found to be required for anaerobic growth, but the absence of norB did not dramatically decrease anaerobic survival. In a wild-type background, induction of norB expression was seen anaerobically in the presence of nitrite but not anaerobically without nitrite or aerobically. norB expression is not regulated by FNR or NarP, but a functional aniA gene (which encodes an anaerobically induced outer membrane nitrite reductase) is necessary for expression. When aniA is constitutively expressed,norB expression can be induced both anaerobically and aerobically, but only in the presence of nitrite, suggesting that nitric oxide, which is likely to be produced by AniA as a product of nitrite reduction, is the inducing agent. This was confirmed with the use of the nitric oxide donor, spermine-nitric oxide complex, in ananiA null background both anaerobically and aerobically. NorB is important for gonococcal adaptation to an anaerobic environment, a physiologically relevant state during gonococcal infection. The presence of this enzyme, which is induced by nitric oxide, may also have implications in immune evasion and immunomodulation in the human host.

scholarly journals The Response ofnorandnosContributes toStaphylococcus aureusVirulence and Metabolism

2019 ◽  
Vol 201 (9) ◽  
Author(s):  
Lacey J. Favazzo ◽  
Ann Lindley Gill ◽  
Christopher W. Farnsworth ◽  
Robert A. Mooney ◽  
Steven R. Gill
Keyword(s):  
Nitric Oxide ◽  
Staphylococcus Aureus ◽  
Reductase Activity ◽  
Wide Spectrum ◽  
Nucleotide Metabolism ◽  
Anaerobic Growth ◽  
Metabolic Adaptation ◽  
Infection Model ◽  
Polymorphonuclear Cells ◽  
Content Type

ABSTRACTStaphylococcus aureuscauses a wide spectrum of disease, with the site and severity of infection dependent on virulence traits encoded within genetically distinct clonal complexes (CCs) and bacterial responses to host innate immunity. The production of nitric oxide (NO) by activated phagocytes is a major host response to whichS. aureusmetabolically adapts through multiple strategies that are conserved in all CCs, including anS. aureusnitric oxide synthase (Nos). Previous genome analysis of CC30, a lineage associated with chronic endocardial and osteoarticular infections, revealed a putative NO reductase (Nor) not found in other CCs that potentially contributes to NO resistance and clinical outcome. Here, we demonstrate that Nor has true nitric oxide reductase activity, withnorexpression enhanced by NO stress and anaerobic growth. Furthermore, we demonstrate thatnoris regulated by MgrA and SrrAB, which modulateS. aureusvirulence and hypoxic response. Transcriptome analysis of theS. aureusUAMS-1, UAMS-1 Δnor, and UAMS-1 Δnosstrains under NO stress and anaerobic growth demonstrates that Nor contributes to nucleotide metabolism and Nos to glycolysis. We demonstrate that Nor and Nos contribute to enhanced survival in the presence of human human polymorphonuclear cells and have organ-specific seeding in a tail vein infection model. Nor contributes to abscess formation in an osteological implant model. We also demonstrate that Nor has a role inS. aureusmetabolism and virulence. The regulation overlap between Nor and Nos points to an intriguing link between regulation of intracellular NO, metabolic adaptation, and persistence in the CC30 lineage.IMPORTANCEStaphylococcus aureuscan cause disease at most body sites, and illness spans asymptomatic infection to death. The variety of clinical presentations is due to the diversity of strains, which are grouped into distinct clonal complexes (CCs) based on genetic differences. The ability ofS. aureusCC30 to cause chronic infections relies on its ability to evade the oxidative/nitrosative defenses of the immune system and survive under different environmental conditions, including differences in oxygen and nitric oxide concentrations. The significance of this work is the exploration of unique genes involved in resisting NO stress and anoxia. A better understanding of the functions that control the response ofS. aureusCC30 to NO and oxygen will guide the treatment of severe disease presentations.

scholarly journals Nitric Oxide Induced stx2 Expression Is Inhibited by the Nitric Oxide Reductase, NorV, in a Clade 8 Escherichia coli O157:H7 Outbreak Strain

2022 ◽  
Vol 10 (1) ◽  
pp. 106
Author(s):  
Rim Al Safadi ◽  
Michelle L. Korir ◽  
Shannon D. Manning
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Shiga Toxin ◽  
Toxin Production ◽  
Anaerobic Growth ◽  
Escherichia Coli O157 ◽  
Similar Increase ◽  
Nitric Oxide Reductase ◽  
Outbreak Strain ◽  
Uremic Syndrome

Escherichia coli O157:H7 pathogenesis is due to Shiga toxin (Stx) production, though variation in virulence has been observed. Clade 8 strains, for instance, were shown to overproduce Stx and were more common among hemolytic uremic syndrome cases. One candidate gene, norV, which encodes a nitric oxide (NO) reductase found in a clade 8 O157:H7 outbreak strain (TW14359), was thought to impact virulence. Hence, we screened for norV in 303 O157 isolates representing multiple clades, examined stx2 expression following NO exposure in TW14359 for comparison to an isogenic mutant (ΔnorV), and evaluated survival in THP-1 derived macrophages. norV was intact in strains representing clades 6–9, whereas a 204 bp deletion was found in clades 2 and 3. During anaerobic growth, NO induced stx2 expression in TW14359. A similar increase in stx2 expression was observed for the ΔnorV mutant in anaerobiosis, though it was not impaired in its ability to survive within macrophages relative to TW14359. Altogether, these data suggest that NO enhances virulence by inducing Stx2 production in TW14359, and that toxin production is inhibited by NorV encoded by a gene found in most clade 8 strains. The mechanism linked to these responses, however, remains unclear and likely varies across genotypes.

scholarly journals Engineering Escherichia coli for Microbial Production of Butanone

2016 ◽  
Vol 82 (9) ◽  
pp. 2574-2584 ◽  
Author(s):  
Kajan Srirangan ◽  
Xuejia Liu ◽  
Lamees Akawi ◽  
Mark Bruder ◽  
Murray Moo-Young ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Batch Cultivation ◽  
Culture Conditions ◽  
Sleeping Beauty ◽  
Genetically Engineered ◽  
Content Type ◽  
Factors Affecting ◽  
Formation Pathway ◽  
E Coli ◽  
Biochemical Genetic

ABSTRACTTo expand the chemical and molecular diversity of biotransformation using whole-cell biocatalysts, we genetically engineered a pathway inEscherichia colifor heterologous production of butanone, an important commodity ketone. First, a 1-propanol-producingE. colihost strain with its sleeping beauty mutase (Sbm) operon being activated was used to increase the pool of propionyl-coenzyme A (propionyl-CoA). Subsequently, molecular heterofusion of propionyl-CoA and acetyl-CoA was conducted to yield 3-ketovaleryl-CoA via a CoA-dependent elongation pathway. Lastly, 3-ketovaleryl-CoA was channeled into the clostridial acetone formation pathway for thioester hydrolysis and subsequent decarboxylation to form butanone. Biochemical, genetic, and metabolic factors affecting relative levels of ketogenesis, acidogenesis, and alcohologenesis under selected fermentative culture conditions were investigated. Using the engineeredE. colistrain for batch cultivation with 30 g liter−1glycerol as the carbon source, we achieved coproduction of 1.3 g liter−1butanone and 2.9 g liter−1acetone. The results suggest that approximately 42% of spent glycerol was utilized for ketone biosynthesis, and thus they demonstrate potential industrial applicability of this microbial platform.

scholarly journals Ralstonia solanacearum Uses Inorganic Nitrogen Metabolism for Virulence, ATP Production, and Detoxification in the Oxygen-Limited Host Xylem Environment

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Beth L. Dalsing ◽  
Alicia N. Truchon ◽  
Enid T. Gonzalez-Orta ◽  
Annett S. Milling ◽  
Caitilyn Allen
Keyword(s):  
Nitric Oxide ◽  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Inorganic Nitrogen ◽  
Anaerobic Growth ◽  
Nitrate Respiration ◽  
Nitrogen Species ◽  
Wild Type ◽  
Content Type ◽  
Xylem Fluid

ABSTRACTGenomic data predict that, in addition to oxygen, the bacterial plant pathogenRalstonia solanacearumcan use nitrate (NO3−), nitrite (NO2−), nitric oxide (NO), and nitrous oxide (N2O) as terminal electron acceptors (TEAs). Genes encoding inorganic nitrogen reduction were highly expressed during tomato bacterial wilt disease, when the pathogen grows in xylem vessels. Direct measurements found that tomato xylem fluid was low in oxygen, especially in plants infected by R. solanacearum. Xylem fluid contained ~25 mM NO3−, corresponding to R. solanacearum's optimal NO3−concentration for anaerobic growthin vitro. We tested the hypothesis that R. solanacearum uses inorganic nitrogen species to respire and grow during pathogenesis by making deletion mutants that each lacked a step in nitrate respiration (ΔnarG), denitrification (ΔaniA, ΔnorB, and ΔnosZ), or NO detoxification (ΔhmpX). TheΔnarG,ΔaniA, andΔnorBmutants grew poorly on NO3−compared to the wild type, and they had reduced adenylate energy charge levels under anaerobiosis. While NarG-dependent NO3−respiration directly enhanced growth, AniA-dependent NO2−reduction did not. NO2−and NO inhibited growth in culture, and their removal depended on denitrification and NO detoxification. Thus, NO3−acts as a TEA, but the resulting NO2−and NO likely do not. None of the mutants grew as well as the wild typein planta, and strains lacking AniA (NO2−reductase) or HmpX (NO detoxification) had reduced virulence on tomato. Thus, R. solanacearum exploits host NO3−to respire, grow, and cause disease. Degradation of NO2−and NO is also important for successful infection and depends on denitrification and NO detoxification systems.IMPORTANCEThe plant-pathogenic bacteriumRalstonia solanacearumcauses bacterial wilt, one of the world's most destructive crop diseases. This pathogen's explosive growth in plant vascular xylem is poorly understood. We used biochemical and genetic approaches to show that R. solanacearum rapidly depletes oxygen in host xylem but can then respire using host nitrate as a terminal electron acceptor. The microbe uses its denitrification pathway to detoxify the reactive nitrogen species nitrite (a product of nitrate respiration) and nitric oxide (a plant defense signal). Detoxification may play synergistic roles in bacterial wilt virulence by converting the host's chemical weapon into an energy source. Mutant bacterial strains lacking elements of the denitrification pathway could not grow as well as the wild type in tomato plants, and some mutants were also reduced in virulence. Our results show how a pathogen's metabolic activity can alter the host environment in ways that increase pathogen success.

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