scholarly journals Improving Arsenic Tolerance of Pyrococcus furiosus by Heterologous Expression of a Respiratory Arsenate Reductase

2020 ◽  
Vol 86 (21) ◽  
Author(s):  
Dominik K. Haja ◽  
Chang-Hao Wu ◽  
Olena Ponomarenko ◽  
Farris L. Poole ◽  
Graham N. George ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Electron Acceptor ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
Arsenate Reductase ◽  
Pyrobaculum Aerophilum ◽  
Terminal Electron Acceptor ◽  
Content Type ◽  
Arsenic Tolerance

ABSTRACT Arsenate is a notorious toxicant that is known to disrupt multiple biochemical pathways. Many microorganisms have developed mechanisms to detoxify arsenate using the ArsC-type arsenate reductase, and some even use arsenate as a terminal electron acceptor for respiration involving arsenate respiratory reductase (Arr). ArsC-type reductases have been studied extensively, but the phylogenetically unrelated Arr system is less investigated and has not been characterized from Archaea. Here, we heterologously expressed the genes encoding Arr from the crenarchaeon Pyrobaculum aerophilum in the euryarchaeon Pyrococcus furiosus, both of which grow optimally near 100°C. Recombinant P. furiosus was grown on molybdenum (Mo)- or tungsten (W)-containing medium, and two types of recombinant Arr enzymes were purified, one containing Mo (Arr-Mo) and one containing W (Arr-W). Purified Arr-Mo had a 140-fold higher specific activity in arsenate [As(V)] reduction than Arr-W, and Arr-Mo also reduced arsenite [As(III)]. The P. furiosus strain expressing Arr-Mo (the Arr strain) was able to use arsenate as a terminal electron acceptor during growth on peptides. In addition, the Arr strain had increased tolerance compared to that of the parent strain to arsenate and also, surprisingly, to arsenite. Compared to the parent, the Arr strain accumulated intracellularly almost an order of magnitude more arsenic when cells were grown in the presence of arsenite. X-ray absorption spectroscopy (XAS) results suggest that the Arr strain of P. furiosus improves its tolerance to arsenite by increasing production of less-toxic arsenate and nontoxic methylated arsenicals compared to that by the parent. IMPORTANCE Arsenate respiratory reductases (Arr) are much less characterized than the detoxifying arsenate reductase system. The heterologous expression and characterization of an Arr from Pyrobaculum aerophilum in Pyrococcus furiosus provides new insights into the function of this enzyme. From in vivo studies, production of Arr not only enabled P. furiosus to use arsenate [As(V)] as a terminal electron acceptor, it also provided the organism with a higher resistance to arsenate and also, surprisingly, to arsenite [As(III)]. In contrast to the tungsten-containing oxidoreductase enzymes natively produced by P. furiosus, recombinant P. aerophilum Arr was much more active with molybdenum than with tungsten. It is also, to our knowledge, the only characterized Arr to be active with both molybdenum and tungsten in the active site.

scholarly journals Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

2015 ◽  
Vol 36 (6) ◽  
pp. 913-922 ◽  
Author(s):  
Nallani Vijay Kumar ◽  
Jianbo Yang ◽  
Jitesh K. Pillai ◽  
Swati Rawat ◽  
Carlos Solano ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Transcriptional Response ◽  
Transcriptional Regulator ◽  
Yeast Protein ◽  
Content Type ◽  
Arsenic Tolerance ◽  
Arsenic Sensing

The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeastSaccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)]in vitroandin vivoand that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

Magnetovibrio blakemorei gen. nov., sp. nov., a magnetotactic bacterium ( Alphaproteobacteria : Rhodospirillaceae ) isolated from a salt marsh

2013 ◽  
Vol 63 (Pt_5) ◽  
pp. 1824-1833 ◽  
Author(s):  
Dennis A. Bazylinski ◽  
Timothy J. Williams ◽  
Christopher T. Lefèvre ◽  
Denis Trubitsyn ◽  
Jiasong Fang ◽  
...  
Keyword(s):  
Salt Marsh ◽  
Electron Acceptor ◽  
Anaerobic Growth ◽  
Rrna Gene ◽  
Magnetotactic Bacterium ◽  
Single Chain ◽  
Circular Chromosome ◽  
Terminal Electron Acceptor ◽  
Content Type ◽  
Link Type

A magnetotactic bacterium, designated strain MV-1T, was isolated from sulfide-rich sediments in a salt marsh near Boston, MA, USA. Cells of strain MV-1T were Gram-negative, and vibrioid to helicoid in morphology. Cells were motile by means of a single polar flagellum. The cells appeared to display a transitional state between axial and polar magnetotaxis: cells swam in both directions, but generally had longer excursions in one direction than the other. Cells possessed a single chain of magnetosomes containing truncated hexaoctahedral crystals of magnetite, positioned along the long axis of the cell. Strain MV-1T was a microaerophile that was also capable of anaerobic growth on some nitrogen oxides. Salinities greater than 10 % seawater were required for growth. Strain MV-1T exhibited chemolithoautotrophic growth on thiosulfate and sulfide with oxygen as the terminal electron acceptor (microaerobic growth) and on thiosulfate using nitrous oxide (N2O) as the terminal electron acceptor (anaerobic growth). Chemo-organoautotrophic and methylotrophic growth was supported by formate under microaerobic conditions. Autotrophic growth occurred via the Calvin–Benson–Bassham cycle. Chemo-organoheterotrophic growth was supported by various organic acids and amino acids, under microaerobic and anaerobic conditions. Optimal growth occurred at pH 7.0 and 26–28 °C. The genome of strain MV-1T consisted of a single, circular chromosome, about 3.7 Mb in size, with a G+C content of 52.9–53.5 mol%.Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MV-1T belongs to the family Rhodospirillaceae within the Alphaproteobacteria , but is not closely related to the genus Magnetospirillum . The name Magnetovibrio blakemorei gen. nov., sp. nov. is proposed for strain MV-1T. The type strain of Magnetovibrio blakemorei is MV-1T ( = ATCC BAA-1436T  = DSM 18854T).

scholarly journals Cytochromes c Constitute a Layer of Protection against Nitric Oxide but Not Nitrite

2018 ◽  
Vol 84 (17) ◽  
Author(s):  
Qiu Meng ◽  
Yijuan Sun ◽  
Haichun Gao
Keyword(s):  
Nitric Oxide ◽  
Specific Activity ◽  
Shewanella Oneidensis ◽  
Main Role ◽  
Content Type ◽  
Environmental Bacterium ◽  
Enzymatic Systems ◽  
Bacteriostatic Agent ◽  
Protective Proteins

ABSTRACT Nitric oxide (NO) is a radical gas that reacts with various biological molecules in complex ways to inhibit growth as a bacteriostatic agent. NO is nearly ubiquitous because it can be generated both biotically and abiotically. To protect the cell from NO damage, bacteria have evolved many strategies, with the production of detoxifying enzymatic systems being the most efficient. Here, we report that c-type cytochromes (cytochromes c) constitute a primary NO protection system in Shewanella oneidensis, a Gram-negative environmental bacterium renowned for respiratory versatility due to its high cytochrome c content. By using mutants producing cytochromes c at varying levels, we found that the content of these proteins is inversely correlated with the growth inhibition imposed by NO, whereas the effect of each individual cytochrome c is negligible. This NO-protecting system has no effect on nitrite inhibition. In the absence of cytochromes c, other NO targets and protective proteins, such as NnrS, emerge to show physiological influences during the NO stress. We further demonstrate that cytochromes c also play a similar role in Escherichia coli, albeit only modestly. Our data thus identify the in vivo function of an important group of proteins in alleviating NO stress. IMPORTANCE It is widely accepted that the antibacterial effects of nitrite are attributable to nitric oxide (NO) formation, suggesting a correlation of bacterial susceptibilities to these two chemicals. However, compared to E. coli, S. oneidensis is highly sensitive to nitrite but resistant to NO, implying the presence of robust NO-protective systems. Here, we show that c-type cytochromes (cytochromes c) play a main role in protecting S. oneidensis against damages from NO but not from nitrite. In their absence, impacts of proteins that promote NO tolerance and that are targets of NO inhibition become evident. Our data thus reveal the specific activity of cytochromes c in alleviating the stress caused by NO but not nitrite.

scholarly journals 6-Hydroxypseudooxynicotine Dehydrogenase Delivers Electrons to Electron Transfer Flavoprotein during Nicotine Degradation by Agrobacterium tumefaciens S33

2019 ◽  
Vol 85 (11) ◽  
Author(s):  
Rongshui Wang ◽  
Jihong Yi ◽  
Jinmeng Shang ◽  
Wenjun Yu ◽  
Zhifeng Li ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Agrobacterium Tumefaciens ◽  
Electron Transport ◽  
Electron Acceptor ◽  
Aromatic Compounds ◽  
Coenzyme Q ◽  
Content Type ◽  
Electron Transfer Flavoprotein ◽  
Nicotine Degradation

ABSTRACT Agrobacterium tumefaciens S33 degrades nicotine via a novel hybrid of the pyridine and the pyrrolidine pathways. The hybrid pathway consists of at least six steps involved in oxidoreductive reactions before the N-heterocycle can be broken down. Collectively, the six steps allow electron transfer from nicotine and its intermediates to the final acceptor O2 via the electron transport chain (ETC). 6-Hydroxypseudooxynicotine oxidase, renamed 6-hydroxypseudooxynicotine dehydrogenase in this study, has been characterized as catalyzing the fourth step using the artificial electron acceptor 2,6-dichlorophenolindophenol. Here, we used biochemical, genetic, and liquid chromatography-mass spectrometry (LC-MS) analyses to determine that 6-hydroxypseudooxynicotine dehydrogenase utilizes the electron transfer flavoprotein (EtfAB) as the physiological electron acceptor to catalyze the dehydrogenation of pseudooxynicotine, an analogue of the true substrate 6-hydroxypseudooxynicotine, in vivo, into 3-succinoyl-semialdehyde-pyridine. NAD(P)+, O2, and ferredoxin could not function as electron acceptors. The oxygen atom in the aldehyde group of the product 3-succinoyl-semialdehyde-pyridine was verified to be derived from H2O. Disruption of the etfAB genes in the nicotine-degrading gene cluster decreased the growth rate of A. tumefaciens S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine, an intermediate downstream of the hybrid pathway, indicating the requirement of EtfAB for efficient nicotine degradation. The electrons were found to be further transferred from the reduced EtfAB to coenzyme Q by the catalysis of electron transfer flavoprotein:ubiquinone oxidoreductase. These results aid in an in-depth understanding of the electron transfer process and energy metabolism involved in the nicotine oxidation and provide novel insights into nicotine catabolism in bacteria. IMPORTANCE Nicotine has been studied as a model for toxic N-heterocyclic aromatic compounds. Microorganisms can catabolize nicotine via various pathways and conserve energy from its oxidation. Although several oxidoreductases have been characterized to participate in nicotine degradation, the electron transfer involved in these processes is poorly understood. In this study, we found that 6-hydroxypseudooxynicotine dehydrogenase, a key enzyme in the hybrid pyridine and pyrrolidine pathway for nicotine degradation in Agrobacterium tumefaciens S33, utilizes EtfAB as a physiological electron acceptor. Catalyzed by the membrane-associated electron transfer flavoprotein:ubiquinone oxidoreductase, the electrons are transferred from the reduced EtfAB to coenzyme Q, which then could enter into the classic ETC. Thus, the route for electron transport from the substrate to O2 could be constructed, by which ATP can be further sythesized via chemiosmosis to support the baterial growth. These findings provide new knowledge regarding the catabolism of N-heterocyclic aromatic compounds in microorganisms.

scholarly journals Intracellular NADPH Levels Affect the Oligomeric State of the Glucose 6-Phosphate Dehydrogenase

2012 ◽  
Vol 11 (12) ◽  
pp. 1503-1511 ◽  
Author(s):  
Michele Saliola ◽  
Angela Tramonti ◽  
Claudio Lanini ◽  
Samantha Cialfi ◽  
Daniela De Biase ◽  
...  
Keyword(s):  
Specific Activity ◽  
Kluyveromyces Lactis ◽  
Growth Conditions ◽  
G6pdh Activity ◽  
Oligomeric State ◽  
Content Type ◽  
Metabolic Role ◽  
Activity Band ◽  
Glucose 6 Phosphate Dehydrogenase

ABSTRACTIn the yeastKluyveromyces lactis, glucose 6-phosphate dehydrogenase (G6PDH) is detected as two differently migrating forms on native polyacrylamide gels. The pivotal metabolic role of G6PDH inK. lactisled us to investigate the mechanism controlling the two activities in respiratory and fermentative mutant strains. An extensive analysis of these mutants showed that the NAD+(H)/NADP+(H)-dependent cytosolic alcohol (ADH) and aldehyde (ALD) dehydrogenase balance affects the expression of the G6PDH activity pattern. Under fermentative/ethanol growth conditions, the concomitant activation of ADH and ALD activities led to cytosolic accumulation of NADPH, triggering an alteration in the oligomeric state of the G6PDH caused by displacement/release of the structural NADP+bound to each subunit of the enzyme. The new oligomeric G6PDH form with faster-migrating properties increases as a consequence of intracellular redox unbalance/NADPH accumulation, which inhibits G6PDH activityin vivo. The appearance of a new G6PDH-specific activity band, following incubation ofSaccharomyces cerevisiaeand human cellular extracts with NADP+, also suggests that a regulatory mechanism of this activity through NADPH accumulation is highly conserved among eukaryotes.

scholarly journals Novel, Oxygen-Insensitive Group 5 [NiFe]-Hydrogenase in Ralstonia eutropha

2013 ◽  
Vol 79 (17) ◽  
pp. 5137-5145 ◽  
Author(s):  
Caspar Schäfer ◽  
Bärbel Friedrich ◽  
Oliver Lenz
Keyword(s):  
Biochemical Characterization ◽  
Ralstonia Eutropha ◽  
Specific Activity ◽  
Atmospheric Oxygen ◽  
Oxidation Activity ◽  
Tetrazolium Chloride ◽  
Content Type ◽  
Activity Measurements ◽  
Group 5

ABSTRACTRecently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H2. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacteriumRalstonia eutrophaH16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by thein vivoH2uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H2. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 μmol of H2per min and mg of protein. However, an unexpectedly high Michaelis constant (Km) for H2of 3.6 ± 0.5 μM was determined, which is in contrast to the previously proposed lowKmof group 5 hydrogenases and makes atmospheric H2uptake byR. eutrophamost unlikely. Amperometric activity measurements revealed that the AH maintains full H2oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O2.

scholarly journals SaeRS Is Responsive to Cellular Respiratory Status and Regulates Fermentative Biofilm Formation in Staphylococcus aureus

2017 ◽  
Vol 85 (8) ◽  
Author(s):  
Ameya A. Mashruwala ◽  
Casey M. Gries ◽  
Tyler D. Scherr ◽  
Tammy Kielian ◽  
Jeffrey M. Boyd
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Protein A ◽  
Regulatory System ◽  
Cellular Respiration ◽  
Signal Molecule ◽  
Terminal Electron Acceptor ◽  
Content Type ◽  
Respiratory Status

ABSTRACT Biofilms are multicellular communities of microorganisms living as a quorum rather than as individual cells. The bacterial human pathogen Staphylococcus aureus uses oxygen as a terminal electron acceptor during respiration. Infected human tissues are hypoxic or anoxic. We recently reported that impaired respiration elicits a programmed cell lysis (PCL) phenomenon in S. aureus leading to the release of cellular polymers that are utilized to form biofilms. PCL is dependent upon the AtlA murein hydrolase and is regulated, in part, by the SrrAB two-component regulatory system (TCRS). In the current study, we report that the SaeRS TCRS also governs fermentative biofilm formation by positively influencing AtlA activity. The SaeRS-modulated factor fibronectin-binding protein A (FnBPA) also contributed to the fermentative biofilm formation phenotype. SaeRS-dependent biofilm formation occurred in response to changes in cellular respiratory status. Genetic evidence presented suggests that a high cellular titer of phosphorylated SaeR is required for biofilm formation. Epistasis analyses found that SaeRS and SrrAB influence biofilm formation independently of one another. Analyses using a mouse model of orthopedic implant-associated biofilm formation found that both SaeRS and SrrAB govern host colonization. Of these two TCRSs, SrrAB was the dominant system driving biofilm formation in vivo. We propose a model wherein impaired cellular respiration stimulates SaeRS via an as yet undefined signal molecule(s), resulting in increasing expression of AtlA and FnBPA and biofilm formation.

scholarly journals Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In Vivo-Like Characteristics

2017 ◽  
Vol 83 (9) ◽  
Author(s):  
Majken Sønderholm ◽  
Kasper Nørskov Kragh ◽  
Klaus Koren ◽  
Tim Holm Jakobsen ◽  
Sophie E. Darch ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Electron Acceptor ◽  
Chronic Wounds ◽  
Alginate Bead ◽  
Alginate Beads ◽  
Model System ◽  
Chronic Infections ◽  
Content Type

ABSTRACT Alginate beads represent a simple and highly reproducible in vitro model system for diffusion-limited bacterial growth. In this study, alginate beads were inoculated with Pseudomonas aeruginosa and followed for up to 72 h. Confocal microscopy revealed that P. aeruginosa formed dense clusters similar in size to in vivo aggregates observed ex vivo in cystic fibrosis lungs and chronic wounds. Bacterial aggregates primarily grew in the bead periphery and decreased in size and abundance toward the center of the bead. Microsensor measurements showed that the O2 concentration decreased rapidly and reached anoxia ∼100 μm below the alginate bead surface. This gradient was relieved in beads supplemented with NO3 − as an alternative electron acceptor allowing for deeper growth into the beads. A comparison of gene expression profiles between planktonic and alginate-encapsulated P. aeruginosa confirmed that the bacteria experienced hypoxic and anoxic growth conditions. Furthermore, alginate-encapsulated P. aeruginosa exhibited a lower respiration rate than the planktonic counterpart and showed a high tolerance toward antibiotics. The inoculation and growth of P. aeruginosa in alginate beads represent a simple and flexible in vivo-like biofilm model system, wherein bacterial growth exhibits central features of in vivo biofilms. This was observed by the formation of small cell aggregates in a secondary matrix with O2-limited growth, which was alleviated by the addition of NO3 − as an alternative electron acceptor, and by reduced respiration rates, as well as an enhanced tolerance to antibiotic treatment. IMPORTANCE Pseudomonas aeruginosa has been studied intensively for decades due to its involvement in chronic infections, such as cystic fibrosis and chronic wounds, where it forms biofilms. Much research has been dedicated to biofilm formation on surfaces; however, in chronic infections, most biofilms form small aggregates of cells not attached to a surface, but embedded in host material. In this study, bacteria were encapsulated in small alginate beads and formed aggregates similar to what is observed in chronic bacterial infections. Our findings show that aggregates are exposed to steep oxygen gradients, with zones of oxygen depletion, and that nitrate may serve as an alternative to oxygen, enabling growth in oxygen-depleted zones. This is important, as slow growth under low-oxygen conditions may render the bacteria tolerant toward antibiotics. This model provides an alternative to surface biofilm models and adds to the comprehension that biofilms do not depend on a surface for formation.

scholarly journals Physiological Adaptation of Desulfitobacterium hafniense Strain TCE1 to Tetrachloroethene Respiration

2011 ◽  
Vol 77 (11) ◽  
pp. 3853-3859 ◽  
Author(s):  
Laure Prat ◽  
Julien Maillard ◽  
Régis Grimaud ◽  
Christof Holliger
Keyword(s):  
Electron Acceptor ◽  
Growth Conditions ◽  
Mass Spectrometry Analysis ◽  
Physiological Adaptation ◽  
Terminal Electron Acceptor ◽  
Organohalide Respiration ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Metabolic Versatility ◽  
Desulfitobacterium Hafniense

ABSTRACTDesulfitobacteriumspp. are ubiquitous organisms with a broad metabolic versatility, and some isolates have the ability to use tetrachloroethene (PCE) as terminal electron acceptor. In order to identify proteins involved in this organohalide respiration process, a comparative proteomic analysis was performed. Soluble and membrane-associated proteins obtained from cells ofDesulfitobacterium hafniensestrain TCE1 that were growing on different combinations of the electron donors lactate and hydrogen and the electron acceptors PCE and fumarate were analyzed. Among proteins increasingly expressed in the presence of PCE compared to fumarate as electron acceptor, a total of 57 proteins were identified by mass spectrometry analysis, revealing proteins involved in stress response and associated regulation pathways, such as PspA, GroEL, and CodY, and also proteins potentially participating in carbon and energy metabolism, such as proteins of the Wood-Ljungdahl pathway and electron transfer flavoproteins. These proteomic results suggest thatD. hafniensestrain TCE1 adapts its physiology to face the relative unfavorable growth conditions during an apparent opportunistic organohalide respiration.

scholarly journals Phenazines Regulate Nap-Dependent Denitrification inPseudomonas aeruginosaBiofilms

2018 ◽  
Vol 200 (9) ◽  
Author(s):  
Yu-Cheng Lin ◽  
Matthew D. Sekedat ◽  
William Cole Cornell ◽  
Gustavo M. Silva ◽  
Chinweike Okegbe ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Nitrate Reductase ◽  
Electron Acceptor ◽  
Bacterial Infections ◽  
Redox State ◽  
Reductase Activity ◽  
Anaerobic Growth ◽  
Bet Hedging ◽  
Terminal Electron Acceptor ◽  
Content Type

ABSTRACTMicrobes in biofilms face the challenge of substrate limitation. In particular, oxygen often becomes limited for cells inPseudomonas aeruginosabiofilms growing in the laboratory or during host colonization. Previously we found that phenazines, antibiotics produced byP. aeruginosa, balance the intracellular redox state of cells in biofilms. Here, we show that genes involved in denitrification are induced in phenazine-null (Δphz) mutant biofilms grown under an aerobic atmosphere, even in the absence of nitrate. This finding suggests that resident cells employ a bet-hedging strategy to anticipate the potential availability of nitrate and counterbalance their highly reduced redox state. Consistent with our previous characterization of aerobically grown colonies supplemented with nitrate, we found that the pathway that is induced in Δphzmutant colonies combines the nitrate reductase activity of the periplasmic enzyme Nap with the downstream reduction of nitrite to nitrogen gas catalyzed by the enzymes Nir, Nor, and Nos. This regulatory relationship differs from the denitrification pathway that functions under anaerobic growth, with nitrate as the terminal electron acceptor, which depends on the membrane-associated nitrate reductase Nar. We identified the sequences in the promoter regions of thenapandniroperons that are required for the effects of phenazines on expression. We also show that specific phenazines have differential effects onnapgene expression. Finally, we provide evidence that individual steps of the denitrification pathway are catalyzed at different depths within aerobically grown biofilms, suggesting metabolic cross-feeding between community subpopulations.IMPORTANCEAn understanding of the unique physiology of cells in biofilms is critical to our ability to treat fungal and bacterial infections. Colony biofilms of the opportunistic pathogenPseudomonas aeruginosagrown under an aerobic atmosphere but without nitrate express a denitrification pathway that differs from that used for anaerobic growth. We report that the components of this pathway are induced by electron acceptor limitation and that they are differentially expressed over the biofilm depth. These observations suggest that (i)P. aeruginosaexhibits “bet hedging,” in that it expends energy and resources to prepare for nitrate availability when other electron acceptors are absent, and (ii) cells in distinct biofilm microniches may be able to exchange substrates to catalyze full denitrification.

Sign in / Sign up

Export Citation Format

Share Document