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Influence of copper on expression of nirS, norB and nosZ and the transcription and activity of NIR, NOR and N2 OR in the denitrifying soil bacteria Pseudomonas stutzeri.

Black A, Hsu PC, Hamonts KE, Clough TJ, Condron LM - Microb Biotechnol (2016)

Bottom Line: Results revealed that 0.05 mM Cu caused maximum conversion of N(2)O to N(2) via bacterial reduction of N(2)O.As soluble Cu generally makes up less than 0.001% of total soil Cu, extrapolation of 0.05 mg l(-l) soluble Cu would require soils to have a total concentration of Cu in the range of, 150-200 μg g(-1) to maximize the proportion of N(2)O reduced to N(2).Given that many intensively farmed agricultural soils are deficient in Cu in terms of plant nutrition, providing a sufficient concentration of biologically accessible Cu could provide a potentially useful microbial-based strategy of reducing agricultural N(2)O emissions.

View Article: PubMed Central - PubMed

Affiliation: Bio Protection Research Centre, Lincoln University, PO Box 85084, Lincoln, Christchurch, 7647, New Zealand.

No MeSH data available.


Related in: MedlinePlus

Expression ratios of the Pseudomonas stutzeri nirS, norB and nosZ at 5 days post‐inoculation. Expression ratios were calculated and normalized against reference genes fdxA, ropD and gyrB. Expression ratios are the difference in gene expression of Pseudomonas stutzeri cultured in basal salt solution with different copper concentration relative to the RNA expression in culture incubated without the presence of copper. Error bars are the SEM for all sample replicates.
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mbt212352-fig-0004: Expression ratios of the Pseudomonas stutzeri nirS, norB and nosZ at 5 days post‐inoculation. Expression ratios were calculated and normalized against reference genes fdxA, ropD and gyrB. Expression ratios are the difference in gene expression of Pseudomonas stutzeri cultured in basal salt solution with different copper concentration relative to the RNA expression in culture incubated without the presence of copper. Error bars are the SEM for all sample replicates.

Mentions: From the Cu treatments it emerges that a Cu concentration of 0.15 mM is the level at which nirS, norB and nosZ expression are at their highest (Fig. 4), although N2O level and the mean N2O:(N2+N2O) ratio was at the highest of all Cu concentration given. Our results show inhibition of nosZ transcription from a Cu concentration of 0.50 mM onwards. Interestingly, even though the nirS enzyme contains a haem‐iron cofactor, it appears the transcription of nirS is responsive to changes in Cu concentrations (Fig. 4). Furthermore, norB expression was also declined with increased Cu concentration. Similarly, in a previous study conducted by Magalhães et al. (2011), a pronounced inhibition by Cu on the transcription of nosZ and nirS was detected. In the treatment containing 0.15 mM of Cu, the N2O:(N2+N2O) ratio was elevated as well as the observation of comparatively high levels of denitrifying genes expressed. This suggests that there could exist an alternative pathway for P. stutzeri to synthesize N2O at higher levels of Cu; thus, the level of N2O produced due to NOR activity could be overestimated in this study. X‐ray crystallographic structures of the periplasmic membrane protein cbb3 cytochrome oxidase of P. stutzeri revealed a high‐spin haem‐copper (CuB) binuclear centre of which the catalytic active site was also found reducing NO to N2O during denitrification process via the proton pathway through K‐channel (Forte et al., 2001; Buschmann et al., 2010). Increased Cu concentration might also contribute in elevating the N2O level via NO reduction by cbb3 cytochrome oxidase in this study. Therefore, expression of genes involved in cbb3 cytochrome oxidase (ccoQNOS) should further be investigated. This may provide a more detailed insight of N2O formation regulated by Cu from an alternative pathway. The enzyme N2OR is considered as the only protein catalysed by Cu due to the multi‐Cu‐sulfide redox centres (Pomowski et al., 2010). Surprisingly, expression of nosZ did not respond to the incensement of Cu concentration in this study, instead, the transcriptional level of nosZ decreased tremendously at 0.50 mM of Cu onward (Fig. 4). This suggested high level of Cu could be displaying inhibitory effect. This result differs with a study conducted by Felgate et al. (2012) where transcription of nosZ of P. denitrificans was found upregulated at high Cu concentration (13 μmol l−1 which is equivalent to 2 mM).


Influence of copper on expression of nirS, norB and nosZ and the transcription and activity of NIR, NOR and N2 OR in the denitrifying soil bacteria Pseudomonas stutzeri.

Black A, Hsu PC, Hamonts KE, Clough TJ, Condron LM - Microb Biotechnol (2016)

Expression ratios of the Pseudomonas stutzeri nirS, norB and nosZ at 5 days post‐inoculation. Expression ratios were calculated and normalized against reference genes fdxA, ropD and gyrB. Expression ratios are the difference in gene expression of Pseudomonas stutzeri cultured in basal salt solution with different copper concentration relative to the RNA expression in culture incubated without the presence of copper. Error bars are the SEM for all sample replicates.
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Related In: Results  -  Collection

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mbt212352-fig-0004: Expression ratios of the Pseudomonas stutzeri nirS, norB and nosZ at 5 days post‐inoculation. Expression ratios were calculated and normalized against reference genes fdxA, ropD and gyrB. Expression ratios are the difference in gene expression of Pseudomonas stutzeri cultured in basal salt solution with different copper concentration relative to the RNA expression in culture incubated without the presence of copper. Error bars are the SEM for all sample replicates.
Mentions: From the Cu treatments it emerges that a Cu concentration of 0.15 mM is the level at which nirS, norB and nosZ expression are at their highest (Fig. 4), although N2O level and the mean N2O:(N2+N2O) ratio was at the highest of all Cu concentration given. Our results show inhibition of nosZ transcription from a Cu concentration of 0.50 mM onwards. Interestingly, even though the nirS enzyme contains a haem‐iron cofactor, it appears the transcription of nirS is responsive to changes in Cu concentrations (Fig. 4). Furthermore, norB expression was also declined with increased Cu concentration. Similarly, in a previous study conducted by Magalhães et al. (2011), a pronounced inhibition by Cu on the transcription of nosZ and nirS was detected. In the treatment containing 0.15 mM of Cu, the N2O:(N2+N2O) ratio was elevated as well as the observation of comparatively high levels of denitrifying genes expressed. This suggests that there could exist an alternative pathway for P. stutzeri to synthesize N2O at higher levels of Cu; thus, the level of N2O produced due to NOR activity could be overestimated in this study. X‐ray crystallographic structures of the periplasmic membrane protein cbb3 cytochrome oxidase of P. stutzeri revealed a high‐spin haem‐copper (CuB) binuclear centre of which the catalytic active site was also found reducing NO to N2O during denitrification process via the proton pathway through K‐channel (Forte et al., 2001; Buschmann et al., 2010). Increased Cu concentration might also contribute in elevating the N2O level via NO reduction by cbb3 cytochrome oxidase in this study. Therefore, expression of genes involved in cbb3 cytochrome oxidase (ccoQNOS) should further be investigated. This may provide a more detailed insight of N2O formation regulated by Cu from an alternative pathway. The enzyme N2OR is considered as the only protein catalysed by Cu due to the multi‐Cu‐sulfide redox centres (Pomowski et al., 2010). Surprisingly, expression of nosZ did not respond to the incensement of Cu concentration in this study, instead, the transcriptional level of nosZ decreased tremendously at 0.50 mM of Cu onward (Fig. 4). This suggested high level of Cu could be displaying inhibitory effect. This result differs with a study conducted by Felgate et al. (2012) where transcription of nosZ of P. denitrificans was found upregulated at high Cu concentration (13 μmol l−1 which is equivalent to 2 mM).

Bottom Line: Results revealed that 0.05 mM Cu caused maximum conversion of N(2)O to N(2) via bacterial reduction of N(2)O.As soluble Cu generally makes up less than 0.001% of total soil Cu, extrapolation of 0.05 mg l(-l) soluble Cu would require soils to have a total concentration of Cu in the range of, 150-200 μg g(-1) to maximize the proportion of N(2)O reduced to N(2).Given that many intensively farmed agricultural soils are deficient in Cu in terms of plant nutrition, providing a sufficient concentration of biologically accessible Cu could provide a potentially useful microbial-based strategy of reducing agricultural N(2)O emissions.

View Article: PubMed Central - PubMed

Affiliation: Bio Protection Research Centre, Lincoln University, PO Box 85084, Lincoln, Christchurch, 7647, New Zealand.

No MeSH data available.


Related in: MedlinePlus