Absence of the Nitrous Oxide Reductase Gene Cluster in Commercial Alfalfa Inoculants Is Probably Due to the Extensive Loss of Genes During Rhizobial Domestication (original) (raw)
Related papers
Microbial Ecology, 2019
We have recently shown that commercial alfalfa inoculants (e.g., Sinorhizobium meliloti B399), which are closely related to the denitrifier model strain Sinorhizobium meliloti 1021, have conserved nitrate, nitrite, and nitric oxide reductases associated with the production of the greenhouse gas nitrous oxide (N 2 O) from nitrate but lost the N 2 O reductase related to the degradation of N 2 O to gas nitrogen. Here, we screened a library of nitrogen-fixing alfalfa symbionts originating from different ecoregions and containing N 2 O reductase genes and identified novel rhizobia (Sinorhizobium meliloti INTA1-6) exhibiting exceptionally low N 2 O emissions. To understand the genetic basis of this novel eco-friendly phenotype, we sequenced and analyzed the genomes of these strains, focusing on their denitrification genes, and found mutations only in the nitrate reductase structural gene napC. The evolutionary analysis supported that, in these natural strains, the denitrification genes were inherited by vertical transfer and that their defective nitrate reductase napC alleles emerged by independent spontaneous mutations. In silico analyses showed that mutations in this gene occurred in ssDNA loop structures with high negative free energy (−ΔG) and that the resulting mutated stem-loop structures exhibited increased stability, suggesting the occurrence of transcription-associated mutation events. In vivo assays supported that at least one of these ssDNA sites is a mutational hot spot under denitrification conditions. Similar benefits from nitrogen fixation were observed when plants were inoculated with the commercial inoculant B399 and strains INTA4-6, suggesting that the low-N 2 O-emitting rhizobia can be an ecological alternative to the current inoculants without resigning economic profitability.
The effect of standard agricultural management on the genetic heterogeneity of nitrous oxide reductase (nosZ) fragments from denitrifying prokaryotes in native and cultivated soil was explored. Thirty-six soil cores were composited from each of the two soil management conditions. nosZ gene fragments were amplified from triplicate samples, and PCR products were cloned and screened by restriction fragment length polymorphism (RFLP). The total nosZ RFLP profiles increased in similarity with soil sample size until triplicate 3-g samples produced visually identical RFLP profiles for each treatment. Large differences in total nosZ profiles were observed between the native and cultivated soils. The fragments representing major groups of clones encountered at least twice and four randomly selected clones with unique RFLP patterns were sequenced to verify nosZ identity. The sequence diversity of nosZ clones from the cultivated field was higher, and only eight patterns were found in clone libraries from both soils among the 182 distinct nosZ RFLP patterns identified from the two soils. A group of clones that comprised 32% of all clones dominated the gene library of native soil, whereas many minor groups were observed in the gene library of cultivated soil. The 95% confidence intervals of the Chao1 nonparametric richness estimator for nosZ RFLP data did not overlap, indicating that the levels of species richness are significantly different in the two soils, the cultivated soil having higher diversity. Phylo-genetic analysis of deduced amino acid sequences grouped the majority of nosZ clones into an interleaved Michigan soil cluster whose cultured members are-Proteobacteria. Only four nosZ sequences from cultivated soil and one from the native soil were related to sequences found in-Proteobacteria. Sequences from the native field formed a distinct, closely related cluster (D mean 0.16) containing 91.6% of the native clones. Clones from the cultivated field were more distantly related to each other (D mean 0.26), and 65% were found outside of the cluster from the native soil, further indicating a difference in the two communities. Overall, there appears to be a relationship between use and richness, diversity, and the phylogenetic position of nosZ sequences, indicating that agricultural use of soil caused a shift to a more diverse denitrifying community. After the last ice age, the nitrous oxide concentration increased in the atmosphere and remained constant (approximately 275 ppb) for about 10,000 years until the 19th century. Since then, the N 2 O concentration has increased significantly to approximately 315 ppb, and this has been predominately attributed to anthropogenic contributions. Due to its long estimated half-life (approximately 120 years), and a global warming potential about 310 times that of carbon dioxide, even a small N 2 O accumulation may cause destructive effects for centuries (4, 46). Denitrification and nitrification are thought to be major sources of atmospheric nitrous oxide (20, 25, 37, 50). The capacity for denitrification is found among a wide variety of taxonomic groups within the Bacteria and Archaea (53). The reduction of nitrous oxide to molecular nitrogen, catalyzed by nitrous oxide reductase, is the last step in the complete deni-trification pathway and represents a respiratory process in its own right, because many denitrifiers can grow at the expense of N 2 O as the sole electron acceptor (28). The plasmid-encoded nature of N 2 O reduction, at least in some strains, distinguishes this respiratory process from other steps in denitrification (43, 54). Numerous environmental factors can vary the proportions of N 2 O and N 2 produced, including soil moisture (3); pH (45); aeration (20, 37); carbon, nitrate, and nitrite availability (34, 45); pore structure (27); and freezing-thawing (25, 36) and drying-wetting (3, 37) events. N 2 O emissions from soils greatly increase with increasing N inputs by fertilization of agricultural soils (10). In most natural habitats, there is usually not enough nitrate to select the large populations of denitrifying organisms. Rather, these denitrifiers are thought to be effective aer-obic competitors for carbon and may seldom use their denitri-fication capacity (45). Lack of carbon as an electron donor almost never prevents denitrification, although it is often not present in amounts that saturate the denitrification capacity. Under starvation conditions, N 2 O was found to be the main product of denitrification in a pure culture of Alcaligenes fae-calis, indicating that an unbalanced supply of electron donor and acceptor may have profound effects on the N 2 O/N 2 ratio (41, 45). Organic matter in soil also serves as a source of nitrate and drives oxygen removal by respiration, creating anaerobic microsites (45). Additionally, differences in oxygen threshold, carbon requirement, and kinetic parameters of various deni
Modularity of nitrogen-oxide reducing soil bacteria: Linking phenotype to genotype
Environmental microbiology, 2016
Model denitrifiers convert NO3 (-) to N2 , but it appears that a significant fraction of natural populations are truncated, conducting only one or two steps of the pathway. To better understand the diversity of partial denitrifiers in soil and whether discrepancies arise between the presence of known N-oxide reductase genes and phenotypic features, bacteria able to reduce NO3 (-) to NO2 (-) were isolated from soil, N-oxide gas products were measured for eight isolates, and six were genome sequenced. Gas phase analyses revealed that two were complete denitrifiers, which genome sequencing corroborated. The remaining six accumulated NO and N2 O to varying degrees and genome sequencing of four indicated that two isolates held genes encoding nitrate reductase as the only dissimilatory N-oxide reductase, one contained genes for both nitrate and nitric oxide reductase, and one had nitrate and nitrite reductase. The results demonstrate that N-oxide production is not always predicted by the ...
Agriculture, Ecosystems & Environment, 2019
While nitrogen fertilizers have helped increase crop yields substantially, they have also contributed to several environmental problems, including an increasing atmospheric concentration of nitrous oxide (N 2 O), a greenhouse gas (GHG) and catalyst of stratospheric ozone decay. The dominant source of atmospheric N 2 O in many agricultural soils is denitrification, a process carried out by soil microbes containing the genes for nitrate reductase (Nar), nitrite reductase (Nir), nitric oxide reductase (Nor) and/or nitrous oxide reductase (Nos). We monitored the abundance of soil nirK, nirS, and nosZ genes during the summer growing season. We sampled replicated field plots from a long-term agricultural research site that includes agroecosystems with corn/soybean/wheat/legume rotations: two tilled-organic systems (Org3 and Org6), and two conventional systems, one using a chisel plow for primary tillage (CT) and one using no-tillage (NT). We demonstrate that nirK copy number in soil was affected primarily by the phase of the crop rotation and secondarily by time of year, regardless of cropping system. In contrast, nosZ gene copy number was primarily driven by cropping system. Soil N 2 O emissions during the sampling period were highest in Org3 and lowest in NT. However, gene quantities did not correspond to N 2 O emissions patterns, indicating that quantitative PCR of key denitrification genes measured at the temporal resolution reported here is not a good predictor of soil N 2 O emissions. These results, nonetheless, show that cropping system management can affect microbial community composition, gene quantity of nir and nos genes and N 2 O emissions. We found cropping system and time of year captured variation in gene abundance among microbial denitrifier populations in these agricultural soils.
2007
Gene sequence analysis of cnorB and qnorB, both encoding nitric oxide reductases, was performed on pure cultures of denitrifiers, for which previously nir genes were analysed. Only 30% of the 227 denitrifying strains rendered a norB amplicon. The cnorB gene was dominant in Alphaproteobacteria, and dominantly coexisted with the nirK gene, coding for the copper-containing nitrite reductase. Both norB genes were equally present in Betaproteobacteria but no linked distributional pattern of nir and norB genes could be observed. The overall cnorB phylogeny was not congruent with the widely accepted organism phylogeny based on 16S rRNA gene sequence analysis, with strains from different bacterial classes having identical cnorB sequences. Denitrifiers and non-denitrifiers could be distinguished through qnorB gene phylogeny, without further grouping at a higher taxonomic resolution. Comparison of nir and norB phylogeny revealed that genetic linkage of both genes is not widespread among denitrifiers. Thus, independent evolution of the genes for both nitrogen oxide reductases does also occur.
Climate, 2018
Nitrous oxide (N2O) is a potent greenhouse gas (GHG). Although it comprises only 0.03% of total GHGs produced, N2O makes a marked contribution to global warming. Much of the N2O in the atmosphere issues from incomplete bacterial denitrification processes acting on high levels of nitrogen (N) in the soil due to fertilizer usage. Using less fertilizer is the obvious solution for denitrification mitigation, but there is a significant drawback (especially where not enough N is available for the crop via N deposition, irrigation water, mineral soil N, or mineralization of organic matter): some crops require high-N fertilizer to produce the yields necessary to help feed the world’s increasing population. Alternatives for denitrification have considerable caveats. The long-standing promise of genetic modification for N fixation may be expanded now to enhance dissimilatory denitrification via genetic engineering. Biotechnology may solve what is thought to be a pivotal environmental challeng...
Reviews in Environmental Science and Bio/Technology, 2020
Nitrous oxide (N 2 O) is a potent greenhouse gas. Agricultural soils are the major source of N 2 O contributing more than 70% of the global N 2 O emissions. In soil, N 2 O is mainly produced during the microbial mediated nitrification and denitrification processes. These processes are influenced by several factors. Nitrification is governed by a few species of bacteria, archaea, and fungi, whereas, several species are involved in the denitrification process. Recently, several improved molecular approaches particularly different omics technologies namely genome sequencing, proteomics, metagenomics, meta-transcriptomics, etc. are being used for microbial diversity assessment, identification of metabolic pathways as well as recovery of the abundant uncultivable microbial genomes along with the genes involved in N 2 O production but also necessary for mitigation of N 2 O Electronic supplementary material The online version of this article (
Isolation, genetic and functional characterization of novel soil nirK-type denitrifiers
2010
Denitrification, the reduction of nitrogen oxides (NO 3 − and NO 2 − ) to N 2 via the intermediates NO and N 2 O, is crucial for nitrogen turnover in soils. Cultivation-independent approaches that applied nitrite reductase genes (nirK/nirS) as marker genes to detect denitrifiers showed a predominance of genes presumably derived from as yet uncultured organisms. However, the phylogenetic affiliation of these organisms remains unresolved since the ability to denitrify is widespread among phylogenetically unrelated organisms. In this study, denitrifiers were cultured using a strategy to generally enrich soil microorganisms. Of 490 colonies screened, eight nirK-containing isolates were phylogenetically identified (16S rRNA genes) as members of the Rhizobiales. A nirK gene related to a large cluster of sequences from uncultured bacteria mainly retrieved from soil was found in three isolates classified as Bradyrhizobium sp. Additional isolates were classified as Bradyrhizobium japonicum and Bosea sp. that contained nirK genes also closely related to the nirK from these strains. These isolates denitrified, albeit with different efficiencies. In Devosia sp., nirK was the only denitrification gene detected. Two Mesorhizobium sp. isolates contained a nirK gene also related to nirK from cultured Mesorhizobia and uncultured soil bacteria but no gene encoding nitric oxide or nitrous oxide reductase. These isolates accumulated NO under nitrate-reducing conditions without growth, presumably due to the lethal effects of NO. This showed the presence of a functional nitrite reductase but lack of a nitric oxide reductase. In summary, similar nirK genotypes recurrently detected mainly in soils likely originated from Rhizobia, and functional differences were presumably strain-dependent.
Applied and Environmental Microbiology, 2005
Transcription of the nirK and nirS genes coding for dissimilatory bacterial nitrite reductases was analyzed by reverse transcription PCR (RT-PCR) of mRNA isolated from rhizosphere samples of three economically important grain legumes at maturity: Vicia faba , Lupinus albus , and Pisum sativum . The nirK gene and transcripts could be detected in all the rhizosphere samples. In contrast, nirS could not be detected. Sampling variations were analyzed by comparing denaturing gradient gel electrophoresis profiles derived from nirK RT-PCR products. High similarity was observed between the replicates, and so one representative product per legume was cloned. Clones with the correct insert size were screened by restriction fragment length polymorphism by using the restriction enzyme MspI. The clones could be distributed into 12 different patterns. Patterns 1, 3, 4, 5, and 7 were common in clone libraries of the three rhizosphere types under study. Patterns 2, 9, 10, and 11 were absent from Pi...