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pH-driven shifts in overall and transcriptionally active denitrifiers control gaseous product stoichiometry in growth experiments with extracted bacteria from soil.

Brenzinger K, Dörsch P, Braker G - Front Microbiol (2015)

Bottom Line: We found that denitrifier community composition, abundance and transcription changed throughout incubation concomitant with pH change in the medium, allowing for complete reduction of nitrate to N2 with little accumulation of intermediates.When exposed to pH 5.4, the denitrifier community was able to grow but reduced N2O to N2 only when near-neutral pH was reestablished by the alkalizing metabolic activity of an acid-tolerant part of the community.Denitrifiers of the nirS-type appeared to be severely suppressed by low pH and nirK-type and nosZ-containing denitrifiers showed strongly reduced transcriptional activity and growth, even after restoration of neutral pH.

View Article: PubMed Central - PubMed

Affiliation: Department of Biogeochemistry, Max Planck Institute for Terrestrial Microbiology Marburg, Germany.

ABSTRACT
Soil pH is a strong regulator for activity as well as for size and composition of denitrifier communities. Low pH not only lowers overall denitrification rates but also influences denitrification kinetics and gaseous product stoichiometry. N2O reductase is particularly sensitive to low pH which seems to impair its activity post-transcriptionally, leading to higher net N2O production. Little is known about how complex soil denitrifier communities respond to pH change and whether their ability to maintain denitrification over a wider pH range relies on phenotypic redundancy. In the present study, we followed the abundance and composition of an overall and transcriptionally active denitrifier community extracted from a farmed organic soil in Sweden (pH H2O = 7.1) when exposed to pH 5.4 and drifting back to pH 6.6. The soil was previously shown to retain much of its functioning (low N2O/N2 ratios) over a wide pH range, suggesting a high functional versatility of the underlying community. We found that denitrifier community composition, abundance and transcription changed throughout incubation concomitant with pH change in the medium, allowing for complete reduction of nitrate to N2 with little accumulation of intermediates. When exposed to pH 5.4, the denitrifier community was able to grow but reduced N2O to N2 only when near-neutral pH was reestablished by the alkalizing metabolic activity of an acid-tolerant part of the community. The genotypes proliferating under these conditions differed from those dominant in the control experiment run at neutral pH. Denitrifiers of the nirS-type appeared to be severely suppressed by low pH and nirK-type and nosZ-containing denitrifiers showed strongly reduced transcriptional activity and growth, even after restoration of neutral pH. Our study suggests that low pH episodes alter transcriptionally active populations which shape denitrifier communities and determine their gas kinetics.

No MeSH data available.


Abundance of functional marker genes for denitrification (nirK, nirS, and nosZ) quantified by qPCR and ratio of cDNA/DNA copy numbers. Left axis, total gene abundance and right axis, ratio of cDNA/DNA copy numbers. Bars indicate the total gene copy numbers and the line the cDNA/DNA ratio. An asterisk indicates significant differences in gene abundance, x indicates significant differences in the ratio of cDNA/DNA copy numbers between incubation at pH 5.4 and pH 7.1 at a given time point (ANOVA: P < 0.05). (A)nirK; (B)nirS; (C)nosZ (Mean±SD, n = 3).
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Figure 3: Abundance of functional marker genes for denitrification (nirK, nirS, and nosZ) quantified by qPCR and ratio of cDNA/DNA copy numbers. Left axis, total gene abundance and right axis, ratio of cDNA/DNA copy numbers. Bars indicate the total gene copy numbers and the line the cDNA/DNA ratio. An asterisk indicates significant differences in gene abundance, x indicates significant differences in the ratio of cDNA/DNA copy numbers between incubation at pH 5.4 and pH 7.1 at a given time point (ANOVA: P < 0.05). (A)nirK; (B)nirS; (C)nosZ (Mean±SD, n = 3).

Mentions: At native pH 7.1, residual O2 after He-washing was depleted and all nitrate was stoichiometrically converted to N2 within 96 h of incubation (Figures 1A,B). Net accumulation of gaseous denitrification intermediates was low (< 0.2% of initially present NO−3-N). Transcriptional activation of functional genes (Figure 2A) and proliferation of denitrifiers containing nirK and nosZ (Figures 3A,C) started instantly after the cells were transferred to the hypoxic medium. A maximum of relative transcription and community size was reached after 96 h (Figures 3A,C), ~40 h after the start of exponential product accumulation (CO2, N2) (Figures 1A,B). The maximum relative transcriptional activity (cDNA/DNA ratio) was low with 0.077 for nirK (Figure 3A) and 0.002 nosZ (Figure 3C), but efficiently translated into denitrifier growth (Figures 3A,C). The strongest growth occurred for nosZ-containing denitrifiers (16,500-fold) while denitrifiers of the nirK-type grew 400-fold (Table S2). In contrast, growth of nirS-type denitrifiers showed a lag-phase of 49 h (Figure 2A, Table S2) after which they were transcriptionally activated (cDNA/DNA ratio of 0.11, Table S3) and increased in abundance, albeit only 50-fold (Figure 3B). Ratios (nosZ/[nirK + nirS]) of >50 after 96 h indicated a tendency of enhanced growth of nosZ-type denitrifiers compared to nitrite reducers (Figure 4, Table S4) which may explain the efficient conversion of N2O to N2 (Philippot et al., 2011). However, PCR-based analyses of genes and transcripts may be biased. The primers used do for instance neither target nirK genotypes from Rhodanobacter species (Green et al., 2010) nor thermophilic Gram-positive denitrifiers (Verbaendert et al., 2014). The recently postulated nosZ clade II (Sanford et al., 2012; Jones et al., 2013) was also not analyzed in this study. Hence, nosZ/(nirK + nirS) ratios and their response to pH must be taken with caution.


pH-driven shifts in overall and transcriptionally active denitrifiers control gaseous product stoichiometry in growth experiments with extracted bacteria from soil.

Brenzinger K, Dörsch P, Braker G - Front Microbiol (2015)

Abundance of functional marker genes for denitrification (nirK, nirS, and nosZ) quantified by qPCR and ratio of cDNA/DNA copy numbers. Left axis, total gene abundance and right axis, ratio of cDNA/DNA copy numbers. Bars indicate the total gene copy numbers and the line the cDNA/DNA ratio. An asterisk indicates significant differences in gene abundance, x indicates significant differences in the ratio of cDNA/DNA copy numbers between incubation at pH 5.4 and pH 7.1 at a given time point (ANOVA: P < 0.05). (A)nirK; (B)nirS; (C)nosZ (Mean±SD, n = 3).
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Figure 3: Abundance of functional marker genes for denitrification (nirK, nirS, and nosZ) quantified by qPCR and ratio of cDNA/DNA copy numbers. Left axis, total gene abundance and right axis, ratio of cDNA/DNA copy numbers. Bars indicate the total gene copy numbers and the line the cDNA/DNA ratio. An asterisk indicates significant differences in gene abundance, x indicates significant differences in the ratio of cDNA/DNA copy numbers between incubation at pH 5.4 and pH 7.1 at a given time point (ANOVA: P < 0.05). (A)nirK; (B)nirS; (C)nosZ (Mean±SD, n = 3).
Mentions: At native pH 7.1, residual O2 after He-washing was depleted and all nitrate was stoichiometrically converted to N2 within 96 h of incubation (Figures 1A,B). Net accumulation of gaseous denitrification intermediates was low (< 0.2% of initially present NO−3-N). Transcriptional activation of functional genes (Figure 2A) and proliferation of denitrifiers containing nirK and nosZ (Figures 3A,C) started instantly after the cells were transferred to the hypoxic medium. A maximum of relative transcription and community size was reached after 96 h (Figures 3A,C), ~40 h after the start of exponential product accumulation (CO2, N2) (Figures 1A,B). The maximum relative transcriptional activity (cDNA/DNA ratio) was low with 0.077 for nirK (Figure 3A) and 0.002 nosZ (Figure 3C), but efficiently translated into denitrifier growth (Figures 3A,C). The strongest growth occurred for nosZ-containing denitrifiers (16,500-fold) while denitrifiers of the nirK-type grew 400-fold (Table S2). In contrast, growth of nirS-type denitrifiers showed a lag-phase of 49 h (Figure 2A, Table S2) after which they were transcriptionally activated (cDNA/DNA ratio of 0.11, Table S3) and increased in abundance, albeit only 50-fold (Figure 3B). Ratios (nosZ/[nirK + nirS]) of >50 after 96 h indicated a tendency of enhanced growth of nosZ-type denitrifiers compared to nitrite reducers (Figure 4, Table S4) which may explain the efficient conversion of N2O to N2 (Philippot et al., 2011). However, PCR-based analyses of genes and transcripts may be biased. The primers used do for instance neither target nirK genotypes from Rhodanobacter species (Green et al., 2010) nor thermophilic Gram-positive denitrifiers (Verbaendert et al., 2014). The recently postulated nosZ clade II (Sanford et al., 2012; Jones et al., 2013) was also not analyzed in this study. Hence, nosZ/(nirK + nirS) ratios and their response to pH must be taken with caution.

Bottom Line: We found that denitrifier community composition, abundance and transcription changed throughout incubation concomitant with pH change in the medium, allowing for complete reduction of nitrate to N2 with little accumulation of intermediates.When exposed to pH 5.4, the denitrifier community was able to grow but reduced N2O to N2 only when near-neutral pH was reestablished by the alkalizing metabolic activity of an acid-tolerant part of the community.Denitrifiers of the nirS-type appeared to be severely suppressed by low pH and nirK-type and nosZ-containing denitrifiers showed strongly reduced transcriptional activity and growth, even after restoration of neutral pH.

View Article: PubMed Central - PubMed

Affiliation: Department of Biogeochemistry, Max Planck Institute for Terrestrial Microbiology Marburg, Germany.

ABSTRACT
Soil pH is a strong regulator for activity as well as for size and composition of denitrifier communities. Low pH not only lowers overall denitrification rates but also influences denitrification kinetics and gaseous product stoichiometry. N2O reductase is particularly sensitive to low pH which seems to impair its activity post-transcriptionally, leading to higher net N2O production. Little is known about how complex soil denitrifier communities respond to pH change and whether their ability to maintain denitrification over a wider pH range relies on phenotypic redundancy. In the present study, we followed the abundance and composition of an overall and transcriptionally active denitrifier community extracted from a farmed organic soil in Sweden (pH H2O = 7.1) when exposed to pH 5.4 and drifting back to pH 6.6. The soil was previously shown to retain much of its functioning (low N2O/N2 ratios) over a wide pH range, suggesting a high functional versatility of the underlying community. We found that denitrifier community composition, abundance and transcription changed throughout incubation concomitant with pH change in the medium, allowing for complete reduction of nitrate to N2 with little accumulation of intermediates. When exposed to pH 5.4, the denitrifier community was able to grow but reduced N2O to N2 only when near-neutral pH was reestablished by the alkalizing metabolic activity of an acid-tolerant part of the community. The genotypes proliferating under these conditions differed from those dominant in the control experiment run at neutral pH. Denitrifiers of the nirS-type appeared to be severely suppressed by low pH and nirK-type and nosZ-containing denitrifiers showed strongly reduced transcriptional activity and growth, even after restoration of neutral pH. Our study suggests that low pH episodes alter transcriptionally active populations which shape denitrifier communities and determine their gas kinetics.

No MeSH data available.