Detection of Nitrate-Reducing/Denitrifying Bacteria from Contaminated and Uncontaminated Tallgrass Prairie Soil: Limitations of PCR Primers
Abstract
:1. Introduction
2. Materials and Methods
2.1. Summary of Sampling Sites and Sampling Protocol
2.2. Selection and Isolation of Nitrate- and Nitrite-Reducing Bacteria
2.3. Obtaining Pure Cultures from Microtiter Plate Wells Positive for DN or NR
2.4. DNA Extraction from Strains
2.5. Molecular Detection of 16S rRNA Gene Sequences and Nitrate Reduction/Denitrification Functional Genes
2.6. DNA Sequencing and Analysis
3. Results
3.1. Distribution of Strains by Origin and Capacity to Reduce Nitrate/Nitrite
3.2. 16S rRNA Phylogenetic Affiliations of the Strains
3.3. Molecular Detection of Nitrate-Reducing Functional Genes napA and narG
3.4. Molecular Detection of Denitrifying Functional Genes nirS, nirK, cnorB, qnorB, and nosZ
4. Discussion
4.1. 16S rRNA Phylogeny and the Reduction of Nitrate/Nitrite
4.2. Considerations When Estimating Numbers of NR and DN Bacteria by PCR
4.3. Long-Term Impact of Brine/Oil Contamination on Nitrogen Cycling in Tallgrass Prairie Soil
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Taxonomic Group | Genus * (Total # of Strains) | Brine/Oil | Oil | Prairie |
---|---|---|---|---|
γ-Proteobacteria | Stenotrophomonas (27) | 3NR ** | 2NR, 3“None” | 11NR, 1DN 7 “None” |
Pseudomonas (9) | 2NR, 2DN | 1NR | 3NR, 1DN | |
Aeromonas (1) | 1NR | 0 | 0 | |
Serratia (1) | 0 | 1NR | 0 | |
Enterobacter (3) | 0 | 3NR | 0 | |
Acinetobacter (5) | 0 | 0 | 2NR, 3 “None” | |
Total # γ Proteobacteria (46) | 8 | 10 | 28 | |
α-Proteobacteria | Ensifer (2) | 1 DN | 0 | 1 DN |
Bosea (2) | 2 “None” | 0 | 0 | |
Phenylobacterium (1) | 1NR | 0 | 0 | |
Total # α- Proteobacteria (5) | 4 | 0 | 1 | |
β-Proteobacteria | Achromobacter (4) | 1NR | 0 | 3NR |
Burkholderia (1) | 0 | 0 | 1 “None” | |
Total # β- Proteobacteria (5) | 1 | 0 | 4 | |
Actinobacteria | Arthrobacter (1) | 1 “None” | 0 | 0 |
Rhodococcus (1) | 0 | 0 | 1 “None” | |
Microbacterium (1) | 0 | 1NR | 0 | |
Kocuria (1) | 0 | 0 | 1NR | |
Total # Actinobacteria (4) | 1 | 1 | 2 | |
Bacilli | Brevibacillus (8) | 3NR | 0 | 4NR, 1“None” |
Bacillus (3) | 2NR | 0 | 1”None” | |
Paenibacillus (1) | 1NR | 0 | 0 | |
Lysinibacillus (2) | 0 | 0 | 1NR, 1”None” | |
Total # Bacilli (14) | 6 | 0 | 8 | |
Flavobacteria | Chryseobacterium (1) | 0 | 1NR | 0 |
Total # Flavobacteria (1) | 0 | 1 | 0 | |
Total # strains isolated | 20 | 12 | 43 | |
Total # NR + DN | 17 | 9 | 28 | |
#NR, #DN | 14, 3 | 9, 0 | 25, 3 |
Taxonomic Group | Genus * (Total # of Strains, # Screened for qnorB, cnorB, nosZ) | NR a | DN b | None c |
---|---|---|---|---|
γ-Proteobacteria | Stenotrophomonas (27, 15) | 16 narG (2) | 1 | 10 |
Pseudomonas (9, 7) | 6 napA (2) | 3 napA (1), nirS (2), cnorB (1), nosZ (1) | 0 | |
Aeromonas (1, 1) | 1 | 0 | 0 | |
Serratia (1, 1) | 1 | 0 | 0 | |
Enterobacter (3, 1) | 3 | 0 | 0 | |
Acinetobacter (5, 1) | 2 | 0 | 3 | |
α-Proteobacteria | Ensifer (2, 1) | 0 | 2 napA (1), cnorB (1) | 0 |
Bosea (2, 0) | 0 | 0 | 2 napA (2) | |
Phenylobacterium (1, 1) | 1 | 0 | 0 | |
β-Proteobacteria | Achromobacter (4, 2) | 4 napA (1) | 0 | 0 |
Burkholderia (1, 0) | 0 | 0 | 1 | |
Actinobacteria | Arthrobacter (1, 0) | 0 | 0 | 1 |
Rhodococcus (1, 0) | 0 | 0 | 1 | |
Microbacterium (1, 1) | 1 | 0 | 0 | |
Kocuria (1, 1) | 1 | 0 | 0 | |
Bacilli | Brevibacillus (8, 1) | 7 | 0 | 1 |
Bacillus (3, 0) | 2 | 0 | 1 | |
Paenibacillus (1, 0) | 1 | 0 | 0 | |
Lysinibacillus (2, 0) | 1 | 0 | 1 | |
Flavobacteria | Chryseobacterium (1, 0) | 1 | 0 | 0 |
Total | 75 (33) | 48 | 6 | 21 |
Strain # (Genus) | Soil Type | napA | narG | nirS | cnorB | nosZ | NR or DN * |
---|---|---|---|---|---|---|---|
1 (Pseudomonas) | Brine/Oil | Yes | Yes | DN | |||
2 (Pseudomonas) | Brine/Oil | Yes | DN | ||||
4 (Ensifer) | Brine/Oil | Yes | DN | ||||
7 (Bosea) | Brine/Oil | Yes | None | ||||
8 (Bosea) | Brine/Oil | Yes | None | ||||
39 (Stenotrophomonas) | Prairie | Yes | NR | ||||
42 (Stenotrophomonas) | Prairie | Yes | NR | ||||
49 (Achromobacter) | Prairie | Yes | NR | ||||
65 (Pseudomonas) | Prairie | Yes | Yes | DN | |||
67 (Pseudomonas) | Prairie | Yes | NR | ||||
68 (Pseudomonas) | Prairie | Yes | NR | ||||
69 (Ensifer) | Prairie | Yes | NR |
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AbuBakr, S.M.; Najar, F.Z.; Duncan, K.E. Detection of Nitrate-Reducing/Denitrifying Bacteria from Contaminated and Uncontaminated Tallgrass Prairie Soil: Limitations of PCR Primers. Microorganisms 2024, 12, 1981. https://doi.org/10.3390/microorganisms12101981
AbuBakr SM, Najar FZ, Duncan KE. Detection of Nitrate-Reducing/Denitrifying Bacteria from Contaminated and Uncontaminated Tallgrass Prairie Soil: Limitations of PCR Primers. Microorganisms. 2024; 12(10):1981. https://doi.org/10.3390/microorganisms12101981
Chicago/Turabian StyleAbuBakr, Samer M., Fares Z. Najar, and Kathleen E. Duncan. 2024. "Detection of Nitrate-Reducing/Denitrifying Bacteria from Contaminated and Uncontaminated Tallgrass Prairie Soil: Limitations of PCR Primers" Microorganisms 12, no. 10: 1981. https://doi.org/10.3390/microorganisms12101981