Nitric Oxide Reductase Gene Expression and Nitrous Oxide Production in Nitrate-Grown Pseudomonas mandelii▿

Pure cultures of Pseudomonas mandelii were incubated with or without nitrate, which acts as a substrate and an electron acceptor for denitrification. Nitric oxide reductase (cnorB) gene expression was measured using a quantitative reverse transcription-PCR, and nitrous oxide emissions were measured by gas chromatography. P. mandelii cells in either the presence or absence of nitrate demonstrated an increase in cnorB gene expression during the first 3 h of growth. The level of expression of cnorB in nitrate-amended cells remained high (average, 2.06 × 108 transcripts/μg of RNA), while in untreated cells it decreased to an average of 3.63 × 106 transcripts/μg of RNA from 4 to 6 h. Nitrous oxide accumulation in the headspace was detected at 2 h, and cumulative emissions continued to increase over a 24-h period to 101 μmol in nitrate-amended cells. P. mandelii cnorB gene expression was not detected under aerobic conditions. These results demonstrate that P. mandelii cnorB gene expression was induced 203-fold at 4 h when nitrate was present in the medium. Accumulations of N2O indicated that the cNorB enzyme was synthesized and active

Effect of Nitrate and Acetylene on nirS, cnorB, and nosZ Expression and Denitrification Activity in Pseudomonas mandelii▿

Nitrate acts as an electron acceptor in the denitrification process. The effect of nitrate in the range of 0 to 1,000 mg/liter on Pseudomonas mandelii nirS, cnorB, and nosZ gene expression was studied, using quantitative reverse transcription-quantitative PCR. Denitrification activity was measured by using the acetylene blockage method and gas chromatography. The effect of acetylene on gene expression was assessed by comparing denitrification gene expression in P. mandelii culture grown in the presence or absence of acetylene. The higher the amount of NO3− present, the greater the induction and the longer the denitrification genes remained expressed. nirS gene expression reached a maximum at 2, 4, 4, and 6 h in cultures grown in the presence of 0, 10, 100, and 1,000 mg of KNO3/liter, respectively, while induction of nirS gene ranged from 12- to 225-fold compared to time zero. cnorB gene expression also followed a similar trend. nosZ gene expression did not respond to NO3− treatment under the conditions tested. Acetylene decreased nosZ gene expression but did not affect nirS or cnorB gene expression. These results showed that nirS and cnorB responded to nitrate concentrations; however, significant denitrification activity was only observed in culture with 1,000 mg of KNO3/liter, indicating that there was no relationship between gene expression and denitrification activity under the conditions tested

Effect of pH and Temperature on Denitrification Gene Expression and Activity in Pseudomonas mandelii▿

Pseudomonas mandelii liquid cultures were studied to determine the effect of pH and temperature on denitrification gene expression, which was quantified by quantitative reverse transcription-PCR. Denitrification was measured by the accumulation of nitrous oxide (N2O) in the headspace in the presence of acetylene. Levels of gene expression of nirS and cnorB at pH 5 were 539-fold and 6,190-fold lower, respectively, than the levels of gene expression for cells grown at pH 6, 7, and 8 between 4 h and 8 h. Cumulative denitrification levels were 28 μmol, 63 μmol, and 22 μmol at pH 6, 7, and 8, respectively, at 8 h, whereas negligible denitrification was measured at pH 5. P. mandelii cells grown at 20°C and 30°C exhibited 9-fold and 94-fold increases in levels of cnorB expression between 0 h and 2 h, respectively, and an average 17-fold increase in levels of nirS gene expression. In contrast, induction of cnorB and nirS gene expression for P. mandelii cells grown at 10°C did not occur in the first 4 h. Levels of cumulative denitrification at 10 h were 6.6 μmol for P. mandelii cells grown at 10°C and 20°C and 30 μmol for cells grown at 30°C. Overall, levels of cnorB and nirS expression were relatively insensitive to pH values over the range of pH 6 to 8 but were substantially reduced at pH 5, whereas gene expression was sensitive to temperature, with induction and time to achieve maximum gene expression delayed as the temperature decreased from 30°C. Low pH and temperature negatively affected denitrification activity