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  • 1
    Language: English
    In: 1215083
    Description: In response to impending anoxic conditions, denitrifying bacteria sustain respiratory metabolism by producing enzymes for reducing nitrogen oxyanions/-oxides (NOx) to N2 (denitrification). Since denitrifying bacteria are non-fermentative, the initial production of denitrification proteome depends on energy from aerobic respiration. Thus, if a cell fails to synthesise a minimum of denitrification proteome before O2 is completely exhausted, it will be unable to produce it later due to energy-limitation. Such entrapment in anoxia is recently claimed to be a major phenomenon in batch cultures of the model organism Paracoccus denitrificans on the basis of measured e−-flow rates to O2 and NOx. Here we constructed a dynamic model and explicitly simulated actual kinetics of recruitment of the cells to denitrification to directly and more accurately estimate the recruited fraction (). Transcription of nirS is pivotal for denitrification, for it triggers a cascade of events leading to the synthesis of a full-fledged denitrification proteome. The model is based on the hypothesis that nirS has a low probability (, h−1) of initial transcription, but once initiated, the transcription is greatly enhanced through positive feedback by NO, resulting in the recruitment of the transcribing cell to denitrification. We assume that the recruitment is initiated as [O2] falls below a critical threshold and terminates (assuming energy-limitation) as [O2] exhausts. With = 0.005 h−1, the model robustly simulates observed denitrification kinetics for a range of culture conditions. The resulting (fraction of the cells recruited to denitrification) falls within 0.038–0.161. In contrast, if the recruitment of the entire population is assumed, the simulated denitrification kinetics deviate grossly from those observed. The phenomenon can be understood as a ‘bet-hedging strategy’: switching to denitrification is a gain if anoxic spell lasts long but is a waste of energy if anoxia turns out to be a ‘false alarm’.
    Keywords: Research Article ; Biology And Life Sciences ; Computer And Information Sciences ; Ecology And Environmental Sciences;
    ISSN: 1553-734X
    E-ISSN: 15537358
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  • 2
    Language: English
    In: Norges forskningsråd: 231282
    Description: Denitrifying bacteria accumulate NO 2 − , NO, and N 2 O, the amounts depending on transcriptional regulation of core denitrification genes in response to O 2 -limiting conditions. The genes include nar , nir , nor and nosZ , encoding NO 3 − -, NO 2 − -, NO- and N 2 O reductase, respectively. We previously constructed a dynamic model to simulate growth and respiration in batch cultures of Paracoccus denitrificans . The observed denitrification kinetics were adequately simulated by assuming a stochastic initiation of nir -transcription in each cell with an extremely low probability (0.5% h -1 ), leading to product- and substrate-induced transcription of nir and nor , respectively, via NO. Thus, the model predicted cell diversification: after O 2 depletion, only a small fraction was able to grow by reducing NO 2 − . Here we have extended the model to simulate batch cultivation with NO 3 − , i.e., NO 2 − , NO, N 2 O, and N 2 kinetics, measured in a novel experiment including frequent measurements of NO 2 − . Pa . denitrificans reduced practically all NO 3 − to NO 2 − before initiating gas production. The NO 2 − production is adequately simulated by assuming stochastic nar -transcription, as that for nirS , but with a higher probability (0.035 h -1 ) and initiating at a higher O 2 concentration. Our model assumes that all cells express nosZ , thus predicting that a majority of cells have only N 2 O-reductase (A), while a minority (B) has NO 2 − -, NO- and N 2 O-reductase. Population B has a higher cell-specific respiration rate than A because the latter can only use N 2 O produced by B. Thus, the ratio B A is low immediately after O 2 depletion, but increases throughout the anoxic phase because B grows faster than A. As a result, the model predicts initially low but gradually increasing N 2 O concentration throughout the anoxic phase, as observed. The modelled cell diversification neatly explains the observed denitrification kinetics and transient intermediate accumulations. The result has major implications for understanding the relationship between genotype and phenotype in denitrification research. Author Summary Denitrifiers generally respire O 2 , but if O 2 becomes limiting, they may switch to anaerobic respiration (denitrification) by producing NO 3 − -, NO 2 − -, NO- and/or N 2 O reductase, encoded by nar , nir , nor , and nosZ genes, respectively. Denitrification causes transient accumulation of NO 2 − and NO/N 2 O emissions, depending on the activity of the four reductases. Denitrifiers lacking nosZ produce ~100% N 2 O, whereas organisms with only nosZ are net consumers of N 2 O. Full-fledged denitrifiers are equipped with all four reductases, genetic regulation of which determines NO 2 − accumulation and NO/N 2 O emissions. Paracoccus denitrificans is a full-fledged denitrifying bacterium, and here we present a modelling approach to understand its gene regulation. We found that the observed transient accumulation of NO 2 − and N 2 O can be neatly explained by assuming cell diversification: all cells expressing nosZ , while a minority expressing nar and nir + nor . Thus, the model predicts that in a batch culture of this organism, only a minor sub-population is full-fledged denitrifier. The cell diversification is a plausible outcome of stochastic initiation of nar- and nir transcription, which then becomes autocatalytic by NO 2 − and NO, respectively. The findings are important for understanding the regulation of denitrification in bacteria: product-induced transcription of denitrification genes is common, and we surmise that diversification in response to anoxia is widespread.
    Keywords: Research Article;
    ISSN: 1553-7358
    ISSN: 1553734X
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  • 3
  • 4
    Language: English
    Description: In response to impending anoxic conditions, denitrifying bacteria sustain respiratory metabolism by producing enzymes for reducing nitrogen oxyanions/-oxides (NOx) to N2 (denitrification). Since denitrifying bacteria are non-fermentative, the initial production of denitrification proteome depends on energy from aerobic respiration. Thus, if a cell fails to synthesise a minimum of denitrification proteome before O2 is completely exhausted, it will be unable to produce it later due to energy-limitation. Such entrapment in anoxia is recently claimed to be a major phenomenon in batch cultures of the model organism Paracoccus denitrificans on the basis of measured e−-flow rates to O2 and NOx. Here we constructed a dynamic model and explicitly simulated actual kinetics of recruitment of the cells to denitrification to directly and more accurately estimate the recruited fraction (). Transcription of nirS is pivotal for denitrification, for it triggers a cascade of events leading to the synthesis of a full-fledged denitrification proteome. The model is based on the hypothesis that nirS has a low probability (, h−1) of initial transcription, but once initiated, the transcription is greatly enhanced through positive feedback by NO, resulting in the recruitment of the transcribing cell to denitrification. We assume that the recruitment is initiated as [O2] falls below a critical threshold and terminates (assuming energy-limitation) as [O2] exhausts. With = 0.005 h−1, the model robustly simulates observed denitrification kinetics for a range of culture conditions. The resulting (fraction of the cells recruited to denitrification) falls within 0.038–0.161. In contrast, if the recruitment of the entire population is assumed, the simulated denitrification kinetics deviate grossly from those observed. The phenomenon can be understood as a ‘bet-hedging strategy’: switching to denitrification is a gain if anoxic spell lasts long but is a waste of energy if anoxia turns out to be a ‘false alarm’.
    Keywords: Vdp::Matematikk Og Naturvitenskap: 400::Basale Biofag: 470::Bioinformatikk: 475 ; Vdp::Mathematics And Natural Scienses: 400::Basic Biosciences: 470::Bioinformatics: 475
    Source: University of Bergen
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  • 5
    Language: English
    Description: -
    Keywords: Vdp::Matematikk Og Naturvitenskap: 400::Basale Biofag: 470::Bioinformatikk: 475 ; Vdp::Mathematics And Natural Scienses: 400::Basic Biosciences: 470::Bioinformatics: 475
    Source: BIBSYS Brage
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  • 6
  • 7
  • 8
    Language: English
    In: Norges forskningsråd: 275389
    Source: Norwegian Open Research Archives (NORA)
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  • 9
    Dissertation
    Dissertation
    Norwegian University of Life Sciences, Ås
    Language: English
    Description: 〈b〉Degree: 〈/b〉Many bacteria respire in the absence of oxygen through reduction of nitrogen oxides (NOx) in the process called denitrification. Denitrification is the main greenhouse gas emitter by its release of N2O when high amounts of N-fertilizers are applied globally. pH is known to be a regulatory factor for N2O emission, but little is known about how quorum sensing regulates denitrification. When respiratory physiology of denitrifying organisms is studied under a defined set of conditions, their phenotypic traits are encompassed by the term denitrification regulatory phenotype (DRP). Pseudomonas aeruginosa is a model organism, well studied due to its widespread denitrifying and opportunistic pathogenic abilities. Detailed gas kinetics of this proteobacterium was studied to characterize its DRP. DRP of strains from P. aeruginosa (a type strain and PAO1 wild type) were characterized with respect to their denitrification phenotype at different initial oxygen concentrations (0, and 7%). This was done by monitoring O2, CO2, NO2-, NO, N2O and N2 by gas chromatography (GC) and an NO-analyzer during their transition from aerobic respiration to denitrification. This study showed that the denser the culture, the higher the accumulation of N2O during denitrification, and implied that quorum sensing (QS) is mediating the N2O emission from denitrification. The question became whether this occurred due to the regulation by one or both of the AHL systems. Further characterization on how QS regulates the denitrification phenotype was done by monitoring denitrification gases under treatment with AHLs on PAO1 rhlI-lasI- mutant and a QS-inhibitor on its PAO1 parent strain (PAO1-UW). Their transcriptional activities of narG, nirS, norB, and nosZ during transition from aerobic respiration to denitrification were quantified by ddPCR. The gas measurements, as well as cell densities, cell numbers and initial biomass were measured to describe specific aerobic and anaerobic respiration rates (μoxic and μanoxic h-1), cell yields per e-acceptor and mRNA per cell. The results showed that the AHL systems´ regulatory effect on denitrification in PAO1 is inhibiting N2OR activity, most likely on a post-transcriptional level. This was directly due to repression of N2O reductase by the Las quorum sensing system.
    Keywords: Quorum Sensing ; Denitrification ; Pseudomonas Aeruginosa
    Source: Norwegian Open Research Archives (NORA)
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  • 10
    Dissertation
    Dissertation
    Norwegian University of Life Sciences, Ås
    Language: English
    Description: Many bacteria respire in the absence of oxygen through reduction of nitrogen oxides (NOx) in the process called denitrification. Denitrification is the main greenhouse gas emitter by its release of N2O when high amounts of N-fertilizers are applied globally. pH is known to be a regulatory factor for N2O emission, but little is known about how quorum sensing regulates denitrification. When respiratory physiology of denitrifying organisms is studied under a defined set of conditions, their phenotypic traits are encompassed by the term denitrification regulatory phenotype (DRP). Pseudomonas aeruginosa is a model organism, well studied due to its widespread denitrifying and opportunistic pathogenic abilities. Detailed gas kinetics of this proteobacterium was studied to characterize its DRP. DRP of strains from P. aeruginosa (a type strain and PAO1 wild type) were characterized with respect to their denitrification phenotype at different initial oxygen concentrations (0, and 7%). This was done by monitoring O2, CO2, NO2-, NO, N2O and N2 by gas chromatography (GC) and an NO-analyzer during their transition from aerobic respiration to denitrification. This study showed that the denser the culture, the higher the accumulation of N2O during denitrification, and implied that quorum sensing (QS) is mediating the N2O emission from denitrification. The question became whether this occurred due to the regulation by one or both of the AHL systems. Further characterization on how QS regulates the denitrification phenotype was done by monitoring denitrification gases under treatment with AHLs on PAO1 rhlI-lasI- mutant and a QS-inhibitor on its PAO1 parent strain (PAO1-UW). Their transcriptional activities of narG, nirS, norB, and nosZ during transition from aerobic respiration to denitrification were quantified by ddPCR. The gas measurements, as well as cell densities, cell numbers and initial biomass were measured to describe specific aerobic and anaerobic respiration rates (μoxic and μanoxic h-1), cell yields per e-acceptor and mRNA per cell. The results showed that the AHL systems´ regulatory effect on denitrification in PAO1 is inhibiting N2OR activity, most likely on a post-transcriptional level. This was directly due to repression of N2O reductase by the Las quorum sensing system.
    Description: Mange bakterier respirerer ved fravær av oksygen gjennom reduksjon av nitrogenoksider (NOx) i en prosess kalt denitrifikasjon. Denitrifikasjon er hoved-klimagass frigjøreren gjennom sitt utslipp av N2O når store mengder N-kunstgjødsel anvendes globalt. pH er en kjent reguleringsfaktor for N2O utslipp, mens quorum sensing (bakterielt kommunikasjonssystem) er en svært lite gjennomsøkt reguleringsmekanisme ved denitrifikasjon. Når respirasjonsfysiologien hos denitrifiserende organismer studeres under definerte forhold, blir de fenotypiske karakterene omfattet av terminen en ”denitrifikasjonsregulatorisk fenotype” (DRP). Pseudomonas aeruginosa er en modellorganisme som er velstudert på grunn av dens utbredte denitrifiserende og opportunistiske patogene egenskaper. Detaljert gasskinetikk av denne proteobakterien ble studert for å karakterisere dens DRP. DRP av stammer fra P. aeruginosa (en type stamme og PAO1 villtypen) ble karakterisert med hensyn til deres denitrifikasjonsfenotype under ulike initielle oksygen konsentrasjoner (0 og 7 %). Dette ble gjort ved overvåkning av O2, CO2, NO2-, NO, N2O og N2 gjennom gasskromatografi (GC) og nitrogen okside-analyse (NOA) under deres overgang fra aerob respirasjon til denitrifikasjon. Dette studiet viste at jo høyere celletettheten var, desto høyere ble N2O akkumuleringen under denitrifikasjon, og antydet at quorum sensing (QS) er medvirkende til N2O utslippet fra denitrifikasjon. Spørsmålet ble om dette skjedde på grunn av reguleringen fra en eller flere AHL systemer. Videre karakterisering av hvordan quorum sensing regulerer denne denitrifikasjonsfenotypen ble gjort ved å overvåke denitrifikasjonsgassene under behandling med ulike AHL på en PAO1 rhIl-lasI- mutant og en QS-hemmer på dens PAO1 forelderstamme. Deres transkripsjons aktivitet av narG, nirS, norB, og nosZ under overgangen fra aerob respirasjon til denitrifikasjon ble kvantifisert ved ddPCR. Gassmålingene, såvel som celletetthet, celletall og initiell biomasse ble målt for å beskrive spesifikk aerob og anaerob respirasjons rate (μoxic and μanoxic h-1), celleutbytte per e- akseptor og mRNA per celle. Resultatene viste at AHL systemenes regulerende effekt på denitrifikasjon i PAO1 hemmer N2OR aktivitet, mest sannsynlig på et post- transkripsjonelt nivå. Dette var direkte på grunn av N2O reduktase-undertrykkelsen ved Las quorum sensing systemet.
    Description: M-MB
    Keywords: N2o ; Quorum Sensing ; Denitrification ; Pseudomonas Aeruginosa ; Mathematics And Natural Science
    Source: BIBSYS Brage
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