RESUMO
The versatile soil bacterium Anaeromyxobacter dehalogenans lacks the hallmark denitrification genes nirS and nirK (encoding NO2-âNO reductases) and couples growth to NO3- reduction to NH4+ (respiratory ammonification) and to N2O reduction to N2A. dehalogenans also grows by reducing Fe(III) to Fe(II), which chemically reacts with NO2- to form N2O (i.e., chemodenitrification). Following the addition of 100 µmol of NO3- or NO2- to Fe(III)-grown axenic cultures of A. dehalogenans, 54 (±7) µmol and 113 (±2) µmol N2O-N, respectively, were produced and subsequently consumed. The conversion of NO3- to N2 in the presence of Fe(II) through linked biotic-abiotic reactions represents an unrecognized ecophysiology of A. dehalogenans The new findings demonstrate that the assessment of gene content alone is insufficient to predict microbial denitrification potential and N loss (i.e., the formation of gaseous N products). A survey of complete bacterial genomes in the NCBI Reference Sequence database coupled with available physiological information revealed that organisms lacking nirS or nirK but with Fe(III) reduction potential and genes for NO3- and N2O reduction are not rare, indicating that NO3- reduction to N2 through linked biotic-abiotic reactions is not limited to A. dehalogenans Considering the ubiquity of iron in soils and sediments and the broad distribution of dissimilatory Fe(III) and NO3- reducers, denitrification independent of NO-forming NO2- reductases (through combined biotic-abiotic reactions) may have substantial contributions to N loss and N2O flux.IMPORTANCE Current attempts to gauge N loss from soils rely on the quantitative measurement of nirK and nirS genes and/or transcripts. In the presence of iron, the common soil bacterium Anaeromyxobacter dehalogenans is capable of denitrification and the production of N2 without the key denitrification genes nirK and nirS Such chemodenitrifiers denitrify through combined biotic and abiotic reactions and have potentially large contributions to N loss to the atmosphere and fill a heretofore unrecognized ecological niche in soil ecosystems. The findings emphasize that the comprehensive understanding of N flux and the accurate assessment of denitrification potential can be achieved only when integrated studies of interlinked biogeochemical cycles are performed.
Assuntos
Proteínas de Bactérias/metabolismo , Desnitrificação , Myxococcales/metabolismo , Nitrito Redutases/deficiência , Microbiologia do Solo , Filogenia , RNA Bacteriano/análise , RNA Ribossômico 16S/análiseRESUMO
Pseudomonas aeruginosa is the major aetiological agent of chronic pulmonary infections in cystic fibrosis (CF) patients. However, recent evidence suggests that the polymicrobial community of the CF lung may also harbour oral streptococci, and colonization by these micro-organisms may have a negative impact on P. aeruginosa within the CF lung. Our previous studies demonstrated that nitrite abundance plays an important role in P. aeruginosa survival during co-infection with oral streptococci. Nitrite reductase is a key enzyme involved in nitrite metabolism. Therefore, the objective of this study was to examine the role nitrite reductase (gene nirS) plays in P. aeruginosa survival during co-infection with an oral streptococcus, Streptococcus parasanguinis. Inactivation of nirS in both the chronic CF isolate FRD1 and acute wound isolate PAO1 reduced the survival rate of P. aeruginosa when co-cultured with S. parasanguinis. Growth of both mutants was restored when co-cultured with S. parasanguinis that was defective for H2O2 production. Furthermore, the nitrite reductase mutant was unable to kill Drosophila melanogaster during co-infection with S. parasanguinis. Taken together, these results suggest that nitrite reductase plays an important role for survival of P. aeruginosa during co-infection with S. parasanguinis.
Assuntos
Nitrito Redutases/deficiência , Nitritos/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus/metabolismo , Animais , Técnicas de Cocultura , Coinfecção/microbiologia , Fibrose Cística/microbiologia , Drosophila melanogaster/microbiologia , Nitrito Redutases/genética , Nitrito Redutases/metabolismo , Pseudomonas aeruginosa/genética , Streptococcus/genéticaRESUMO
The Neurospora crassa mutants nit-2 (lacking both nitrite and nitrate reductases) and nit-6 (lacking nitrite reductase) grown in the medium with ammonium chloride as a sole source of nitrogen discharged nitrate and nitrite ions into culture medium. For nit-2, the content of nitrate exceeded that of nitrite in both the homogenate of fungal cells and growth medium; moreover, this difference was more pronounced in the culture medium. Unlike nit-2, the content of nitrite in the cultivation medium of the nit-6 mutant irradiated with visible light for 30 min during the lag phase of carotenogenesis photoinduction displayed a trend of increase as compared with the dark control. Further (to 240 min) irradiation of cells, i.e., irradiation during biosynthesis of carotenoid pigments, leveled this difference.
Assuntos
Mutação , Neurospora crassa/metabolismo , Nitrato Redutase/deficiência , Nitratos/metabolismo , Nitrito Redutases/deficiência , Nitritos/metabolismo , Cloreto de Amônio/metabolismo , Cloreto de Amônio/farmacologia , Carotenoides , Proteínas de Ligação a DNA/deficiência , Proteínas Fúngicas , Neurospora crassa/genética , Fatores de Transcrição/deficiênciaRESUMO
Pseudomonas putida JLR11 can grow on 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. We created nasB (nitrite reductase), pnrA (nitroaromatic reductase) and pnrA nasB mutants and tested their growth with TNT as the sole N source. The nasB and pnrA mutants grew at a reduced rate on TNT, whereas the double nasB pnrA mutant did not. This suggests that P. putida JLR11 carries out multiple enzymatic attacks on TNT-releasing nitrite and/or ammonium. The PnrA nitroreductase plays a key role in the reduction of TNT to 2,6-dinitro-4-hydroxylaminotoluene and the subsequent release of ammonium for growth.
Assuntos
Nitrito Redutases/deficiência , Nitrorredutases/deficiência , Pseudomonas putida/enzimologia , Trinitrotolueno/metabolismo , Nitrito Redutases/genética , Nitrorredutases/genética , Pseudomonas putida/genética , Pseudomonas putida/crescimento & desenvolvimentoRESUMO
A gene that encodes a periplasmic copper-type nitrite reductase (NirK) was identified in Nitrosomonas europaea. Disruption of this gene resulted in the disappearance of Nir activity in cell extracts. The nitrite tolerance of NirK-deficient cells was lower than that of wild-type cells. Unexpectedly, NirK-deficient cells still produced nitric oxide (NO) and nitrous oxide (N(2)O), the latter in greater amounts than that of wild-type cells. This demonstrates that NirK is not essential for the production of NO and N(2)O by N. europaea. Inactivation of the putative fnr gene showed that Fnr is not essential for the expression of nirK.
Assuntos
Proteínas de Escherichia coli , Nitrito Redutases/metabolismo , Nitritos/metabolismo , Óxidos de Nitrogênio/metabolismo , Nitrosomonas/enzimologia , Proteínas de Bactérias/genética , Deleção de Genes , Expressão Gênica , Proteínas Ferro-Enxofre/deficiência , Proteínas Ferro-Enxofre/genética , Nitrito Redutases/deficiência , Nitrito Redutases/genética , Nitrosomonas/genética , Óxido Nitroso/metabolismo , Transcrição GênicaRESUMO
We constructed mutant strains lacking the nitrite reductase (NR) gene in Chlamydomonas reinhardtii. Two types of NR mutants were obtained, which either have or lack the high-affinity nitrate transporter (Nrt2;1, Nrt2;2, and Nar2) genes. None of these mutants overexpressed nitrate assimilation gene transcripts nor NR activity in nitrogen-free medium, in contrast to NR mutants. This finding confirms the previous role proposed for NR on its own regulation (autoregulation) and on the other genes for nitrate assimilation in C. reinhardtii. In addition, the NR mutants were used to study nitrate transporters from nitrite excretion. At high CO(2), only strains carrying the above high-affinity nitrate transporter genes excreted stoichiometric amounts of nitrite from 100 microM nitrate in the medium. A double mutant, deficient in both the high-affinity nitrate transporter genes and NR, excreted nitrite at high CO(2) only when nitrate was present at mM concentrations. This suggests that there exists a low-affinity nitrate transporter that might correspond to the nitrate/nitrite transport system III. Moreover, under low CO(2) conditions, the double mutant excreted nitrite from nitrate at micromolar concentrations by a transporter with the properties of the nitrate/nitrite transport system IV.
Assuntos
Proteínas de Transporte de Ânions , Proteínas de Transporte/genética , Chlamydomonas reinhardtii/genética , Nitratos/metabolismo , Nitrito Redutases/genética , Proteínas Nucleares/genética , Proteínas de Plantas , Proteínas de Protozoários , Animais , Transporte Biológico , Western Blotting , Proteínas de Transporte/metabolismo , Chlamydomonas reinhardtii/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Transportadores de Nitrato , Nitrito Redutases/deficiência , Nitrito Redutases/metabolismo , Proteínas Nucleares/metabolismoRESUMO
Anaerobic, but not aerobic, cultures of Escherichia coli K-12 catalysed the rapid nitrosation of the model substrate 2,3-diaminonaphthalene when incubated with nitrite. Formate and lactate were effective electron donors for the nitrosation reaction, which was inhibited by nitrate. Optimal growth conditions for the expression of nitrosation activity by various strains and mutants were determined. Highest activities were found with bacteria that had been grown anaerobically in a minimal medium rather than in Lennox broth, with glycerol and fumarate rather than glucose as the main carbon and energy source, and in the presence of a low concentration of nitrate. Bacteria harvested in the early exponential phase were more active than those harvested in later stages of growth. Well-characterized mutants defective in the synthesis of one or more anaerobically induced electron transfer chains were screened for nitrosation activity under these optimal growth conditions: only the respiratory nitrate reductase encoded by the narGHJI operon was implicated as a major contributor to nitrosation activity. Due to the limited sensitivity of the assays currently available, a minor contribution from the two alternative nitrate reductases or even other molybdoproteins could not be excluded. The role of formate in nitrosation was complex and was clearly not limited simply to that of an electron donor in the bacterial reduction of nitrite to nitric oxide: at least two further, chemical roles were inferred. This extensive study of more than 400 independent cultures of E. coli K-12 and its derivatives resolved some, but not all, of the apparently conflicting data in the literature concerning nitrosation catalysed by enteric bacteria.
Assuntos
Escherichia coli/genética , Nitritos/metabolismo , Compostos Nitrosos/metabolismo , 2-Naftilamina/análogos & derivados , 2-Naftilamina/metabolismo , Aerobiose , Anaerobiose , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Formiatos/metabolismo , Mutação , Nitrato Redutase , Nitrato Redutases/deficiência , Nitrato Redutases/genética , Nitratos/farmacologia , Nitrito Redutases/deficiência , Nitrito Redutases/genética , OxirreduçãoRESUMO
Two gentamicin-resistant mutants of Pseudomonas aeruginosa PAO 503 were selected after ethyl methane sulfonate mutagenesis. Mutant PAO 2403 had significantly increased resistance to aminoglycoside but not to other antibiotics. Mutant PAO 2402 showed a similar spectrum of resistance but of lower magnitude. Both mutants showed no detectable cytochrome d and had a high frequency of reversion to a fully wild-type phenotype. PAO 2403 had a marked decrease and PAO 2402 had a moderate decrease in nitrite reductase activity. Both mutants had reduced uptake of gentamicin and dihydrostreptomycin. Mutant PAO 2403 showed a general decrease in transport rate of cationic compounds, whereas mutant PAO 2402 had only deficient glucose transport. Both mutants showed enhanced rates of glutamine transport and no change in glutamic acid transport. Other components of electron transport and oxidative phosphorylation were normal. These mutants involve ferrocytochrome C551 oxidoreductase formed only on anaerobic growth but illustrate transport defects in aerobically grown cells.