Bacteria rather than Archaea dominate microbial ammonia oxidation in an agricultural soil

Agricultural ecosystems annually receive approximately 25% of the global nitrogen input, much of which is oxidized at least once by ammonia-oxidizing prokaryotes to complete the nitrogen cycle. Recent discoveries have expanded the known ammonia-oxidizing prokaryotes from the domain Bacteria to Archaea . However, in the complex soil environment it remains unclear whether ammonia oxidation is exclusively or predominantly linked to Archaea as implied by their exceptionally high abundance. Here we show that Bacteria rather than Archaea functionally dominate ammonia oxidation in an agricultural soil, despite the fact that archaeal versus bacterial amoA genes are numerically more dominant. In soil microcosms, in which ammonia oxidation was stimulated by ammonium and inhibited by acetylene, activity change was paralleled by abundance change of bacterial but not of archaeal amoA gene copy numbers. Molecular fingerprinting of amoA genes also coupled ammonia oxidation activity with bacterial but not archaeal amoA gene patterns. DNA-stable isotope probing demonstrated CO₂ assimilation by Bacteria rather than Archaea . Our results indicate that Archaea were not important for ammonia oxidation in the agricultural soil tested.     

Ammonia-oxidizing archaea have more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils

Increasing evidence demonstrated the involvement of ammonia-oxidizing archaea (AOA) in the global nitrogen cycle, but the relative contributions of AOA and ammonia-oxidizing bacteria (AOB) to ammonia oxidation are still in debate. Previous studies suggest that AOA would be more adapted to ammonia-limited oligotrophic conditions, which seems to be favored by protonation of ammonia, turning into ammonium in low-pH environments. Here, we investigated the autotrophic nitrification activity of AOA and AOB in five strongly acidic soils (pH<4.50) during microcosm incubation for 30 days. Significantly positive correlations between nitrate concentration and amoA gene abundance of AOA, but not of AOB, were observed during the active nitrification. ¹³CO₂-DNA-stable isotope probing results showed significant assimilation of ¹³C-labeled carbon source into the amoA gene of AOA, but not of AOB, in one of the selected soil samples. High levels of thaumarchaeal amoA gene abundance were observed during the active nitrification, coupled with increasing intensity of two denaturing gradient gel electrophoresis bands for specific thaumarchaeal community. Addition of the nitrification inhibitor dicyandiamide (DCD) completely inhibited the nitrification activity and CO₂ fixation by AOA, accompanied by decreasing thaumarchaeal amoA gene abundance. Bacterial amoA gene abundance decreased in all microcosms irrespective of DCD addition, and mostly showed no correlation with nitrate concentrations. Phylogenetic analysis of thaumarchaeal amoA gene and 16S rRNA gene revealed active ¹³CO₂-labeled AOA belonged to groups 1.1a-associated and 1.1b. Taken together, these results provided strong evidence that AOA have a more important role than AOB in autotrophic ammonia oxidation in strongly acidic soils.     

Metagenomic and network analyses reveal key players in nitrification in upland agricultural soils

Nitrification, a key step in soil nitrogen cycling, is a biologically mediated process crucial to the ecological environment. However, how nitrifiers drive nitrification under different soil properties and climatic factors at large spatial scales is poorly understood. Here, using metagenomic sequencing and network-based approaches, we identified key nitrifying species of upland agricultural soils in northern China, which spans a wide range of climates and geographic distances. We found that potential nitrification rates (PNRs) varied in different soils and were positively correlated with soil pH (5.42–8.46) and mean annual temperature (MAT) and negatively correlated with the C/N ratio. Network analysis revealed that one module (module 3) was significantly correlated with PNR. In this module, 16 dominant nodes were associated with AOB Nitrosomonas and most nodes were significantly correlated with environmental factors, suggesting that abiotic conditions are important for determining the assembly of these key nitrifiers. Our study advanced the understanding of the key nitrifying populations and their environmental drivers in upland agricultural soil across different soil and climate types.     

Plasmid incidence in bacteria from agricultural and industrial soils

Heavy metal contents of agricultural and industrial soils were determined by atomic absorption spectrophotometry. The analysis of the samples collected from two different locations revealed significantly high levels of Fe, Zn, Cu, Cr and Ni. Certain microbiological parameters (total aerobic heterotrophs, asymbiotic N₂-fixers, total Actinomycetes and fungi) were also monitored from these soils. A total of 70 bacterial isolates from agricultural and industrial soils were examined for plasmid DNA content and resistance to the antibiotics amoxycillin, cloxacillin, chloramphenicol, doxycycline methicillin, nalidixic acid, and tetracycline. Minimum inhibitory concentrations (MICs) of Cu, Cr, Pb, Cd, Hg, Zn, and Ni for each isolate were also determined. Resistance was most frequent to methicillin (48.5%), cloxacillin (45.7%), and nalidixic acid (40%) for all isolates of bacteria. The highest MICs observed were 100 g/ml for mercury, 800 g/ml for Ni and 1600 g/ml for other metals. The incidences of metal resistance and MICs of metals for bacteria from industrial soil were significantly different to those of agricultural soil. On a percentage basis, 91.4% of the total bacterial isolates from industrial soil were found to harbour plasmids whereas 40% of the isolates from agricultural soil contained plasmids.     

Climate rather than geography separates two European honeybee subspecies

Both climatic and geographical factors play an important role for the biogeographical distribution of species. The Carpathian mountain ridge has been suggested as a natural geographical divide between the two honeybee subspecies Apis mellifera carnica and A. m. macedonica. We sampled one worker from one colony each at 138 traditional apiaries located across the Carpathians spanning from the Hungarian plains to the Danube delta. All samples were sequenced at the mitochondrial tRNA^Leu‐cox2 intergenic region and genotyped at twelve microsatellite loci. The Carpathians had only limited impact on the biogeography because both subspecies were abundant on either side of the mountain ridge. In contrast, subspecies differentiation strongly correlated with the various temperature zones in Romania. A. m. carnica is more abundant in regions with the mean average temperature below 9 °C, whereas A. m. macedonica honeybees are more frequent in regions with mean temperatures above 9 °C. This range selection may have impact on the future biogeography in the light of anticipated global climatic changes.     

Links between ammonia oxidizer community structure, abundance, and nitrification potential in acidic soils

Ammonia oxidation is the first and rate-limiting step of nitrification and is performed by both ammonia-oxidizing archaea (AOA) and bacteria (AOB). However, the environmental drivers controlling the abundance, composition, and activity of AOA and AOB communities are not well characterized, and the relative importance of these two groups in soil nitrification is still debated. Chinese tea orchard soils provide an excellent system for investigating the long-term effects of low pH and nitrogen fertilization strategies. AOA and AOB abundance and community composition were therefore investigated in tea soils and adjacent pine forest soils, using quantitative PCR (qPCR), terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis of respective ammonia monooxygenase (amoA) genes. There was strong evidence that soil pH was an important factor controlling AOB but not AOA abundance, and the ratio of AOA to AOB amoA gene abundance increased with decreasing soil pH in the tea orchard soils. In contrast, T-RFLP analysis suggested that soil pH was a key explanatory variable for both AOA and AOB community structure, but a significant relationship between community abundance and nitrification potential was observed only for AOA. High potential nitrification rates indicated that nitrification was mainly driven by AOA in these acidic soils. Dominant AOA amoA sequences in the highly acidic tea soils were all placed within a specific clade, and one AOA genotype appears to be well adapted to growth in highly acidic soils. Specific AOA and AOB populations dominated in soils at particular pH values and N content, suggesting adaptation to specific niches.     

Selenoproteins in Archaea and Gram-positive bacteria

Selenium is an essential trace element for many organisms by serving important catalytic roles in the form of the 21st co-translationally inserted amino acid selenocysteine. It is mostly found in redox-active proteins in members of all three domains of life and analysis of the ever-increasing number of genome sequences has facilitated identification of the encoded selenoproteins. Available data from biochemical, sequence, and structure analyses indicate that Gram-positive bacteria synthesize and incorporate selenocysteine via the same pathway as enterobacteria. However, recent in vivo studies indicate that selenocysteine-decoding is much less stringent in Gram-positive bacteria than in Escherichia coli. For years, knowledge about the pathway of selenocysteine synthesis in Archaea and Eukarya was only fragmentary, but genetic and biochemical studies guided by analysis of genome sequences of Sec-encoding archaea has not only led to the characterization of the pathways but has also shown that they are principally identical. This review summarizes current knowledge about the metabolic pathways of Archaea and Gram-positive bacteria where selenium is involved, about the known selenoproteins, and about the respective pathways employed in selenoprotein synthesis.     

长期施加氮肥及氧化钙调节对酸性土壤硝化作用及氨氧化微生物的影响

以长期施加氮肥及添加氧化钙调节的酸性土壤为研究对象,运用定量PCR和DGGE技术,探讨了土壤氨氧化微生物及硝化作用对不同施肥处理及氧化钙调节的响应。长期施化学氮肥导致酸性土壤p H(KCl)值(3.35—3.47)和硝化潜势(0.02—0.14μg NO-2-N g-1土壤h-1)进一步降低,而添加Ca O后土壤酸化得以缓解(p H值4.10—4.46),土壤硝化潜势(0.22—0.34μg NO-2-N g-1土h-1)显著增加。同时,添加Ca O处理对氨氧化古菌(AOA)的群落结构无明显影响,但明显提高了各施肥处理土壤中氨氧化细菌(AOB)的群落多样性,加Ca O处理的土壤中,AOA的数量降低而AOB的数量增加。这些结果表明虽然酸性土壤中AOA在数量和活性上占主导优势,AOB在功能上冗余,但当添加Ca O后,AOA和AOB对环境变化迅速作出响应,并根据其不同的生态位需求重新分配优势地位,二者交替作用共同驱动酸性土壤硝化作用。     

Urease gene‐containing Archaea dominate autotrophic ammonia oxidation in two acid soils

The metabolic traits of ammonia‐oxidizing archaea (AOA) and bacteria (AOB) interacting with their environment determine the nitrogen cycle at the global scale. Ureolytic metabolism has long been proposed as a mechanism for AOB to cope with substrate paucity in acid soil, but it remains unclear whether urea hydrolysis could afford AOA greater ecological advantages. By combining DNA‐based stable isotope probing (SIP) and high‐throughput pyrosequencing, here we show that autotrophic ammonia oxidation in two acid soils was predominately driven by AOA that contain ureC genes encoding the alpha subunit of a putative archaeal urease. In urea‐amended SIP microcosms of forest soil (pH 5.40) and tea orchard soil (pH 3.75), nitrification activity was stimulated significantly by urea fertilization when compared with water‐amended soils in which nitrification resulted solely from the oxidation of ammonia generated through mineralization of soil organic nitrogen. The stimulated activity was paralleled by changes in abundance and composition of archaeal amoA genes. Time‐course incubations indicated that archaeal amoA genes were increasingly labelled by ¹³CO₂ in both microcosms amended with water and urea. Pyrosequencing revealed that archaeal populations were labelled to a much greater extent in soils amended with urea than water. Furthermore, archaeal ureC genes were successfully amplified in the ¹³C‐DNA, and acetylene inhibition suggests that autotrophic growth of urease‐containing AOA depended on energy generation through ammonia oxidation. The sequences of AOB were not detected, and active AOA were affiliated with the marine Group 1.1a‐associated lineage. The results suggest that ureolytic N metabolism could afford AOA greater advantages for autotrophic ammonia oxidation in acid soil, but the mechanism of how urea activates AOA cells remains unclear.     

Diversity of Archaea in Brazilian savanna soils

Although the richness of Bacteria and Fungi in Cerrado’ soils has been reported, here we report, for the first time, the archaeal community in Cerrado’s soils. DNA extracted from soil of two distinct vegetation types, a dense subtype of sensu strict (cerrado denso) and riverbank forest (mata de galeria), was used to amplify Archaea-specific 16S rRNA gene. All of the fragments sequenced were classified as Archaea into the phylum Thaumarchaeota, predominantly affiliated to groups I.1b and I.1c. Sequences affiliated to the group I.1a were found only in the soil from riverbank forest. Soils from ‘cerrado denso’ had greater Archaea richness than those from ‘mata de galeria’ based on the richness indexes and on the rarefaction curve. β-Diversity analysis showed significant differences between the sequences from the two soil areas studied because of their different thaumarchaeal group composition. These results provide information about the third domain of life from Cerrado soils.     

Heterotrophic nitrification in add forest soils

Ninety cultures of heterotrophic organisms were isolated from soils of four acid Norwegian forest sites, which were active in nitrifying. The isolates were tested for ability to form nitrite in a glucose-ammonium-inorganic salts medium. Eleven cultures were found capable of oxidizing ammonium to nitrite. The nitrifying organisms consist of 4 bacteria and 7 fungi.     

Genetic and phenotypic diversity of carbofuran-degrading bacteria isolated from agricultural soils

Thirty-seven carbofuran-degrading bacteria were isolated from agricultural soils, and their genetic and phenotypic characteristics were investigated. The isolates were able to utilize carbofuran as a sole source of carbon and energy. Analysis of the 16S rRNA gene sequence indicated that the isolates were related to members of the genera Rhodococcus, Sphingomonas, and Sphingobium, including new types of carbofuran-degrading bacteria, Bosea and Microbacterium. Among the 37 isolates, 15 different chromosomal DNA patterns were obtained by polymerase chain reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences. Five of the 15 representative isolates were able to degrade carbofuran phenol, fenoxycarb, and carbaryl, in addition to carbofuran. Ten of the 15 representative isolates had 1 to 8 plasmids. Among the 10 plasmid-containing isolates, plasmid-cured strains were obtained from 5 strains. The cured strains could not degrade carbofuran and other pesticides anymore, suggesting that the carbofuran degradative genes were on the plasmid DNAs in these strains. When analyzed with PCR amplification and dot-blot hybridization using the primers targeting for the previously reported carbofuran hydrolase gene (mcd), all of the isolates did not show any positive signals, suggesting that their carbofuran hydrolase genes had no significant sequence homology with the mcd gene.     

Dynamics and functional relevance of ammonia-oxidizing archaea in two agricultural soils

Crucial steps in geochemical cycles are in many cases performed by more than one group of microorganisms, but the significance of this functional redundancy with respect to ecosystem functioning is poorly understood. Ammonia-oxidizing archaea (AOA) and their bacterial counterparts (AOB) are a perfect system to address this question: although performing the same transformation step, they belong to well-separated phylogenetic groups. Using pig manure amended with different concentrations of sulfadiazine (SDZ), an antibiotic that is frequently used in veterinary medicine, it was possible to affect AOB and AOA to different degrees. Addition of manure stimulated growth of AOB in both soils and, interestingly, also growth of AOA was considerably stimulated in one of the soils. The antibiotic treatments decreased the manure effect notably on AOB, whereas AOA were affected to a lower extent. Model calculations concerning the respective proportions of AOA and AOB in ammonia oxidation indicate a substantial contribution of AOA in one of the soils that further increased under the influence of SDZ, hence indicating functional redundancy between AOA and AOB.     

Estimation of nitrification rates in flooded soils

Three techniques for estimating nitrification rates in flooded soils were evaluated in short-term incubation experiments using three soils. The techniques were based on inhibition of either ammonium or nitrite oxidation and ¹³N isotope dilution. Of four inhibitors of ammonium oxidation evaluated, one (allylthiourea) was ineffective and two (2-ethynylpyridine or phenyl acetylene dissolved in ethanol) promoted immobilization of ammonium. Emulsified 2-ethynylpyridine and acetylene were equally effective inhibitors of ammonium oxidation and had little or no effect on gross rates of N mineralization and immobilization. Four inhibitors of nitrite oxidation were evaluated, but this approach was compromised by the nonspecificity of three of the compounds—potassium cyanide, 2-ethylamino-4-isopropylamino-6-methylthio-s-triazine (ametryne) and 3-(3,4-dichlorophenyl)-1-methylurea (DMU)—and by the partial effectiveness of another (potassium chlorate). Two methods based on isotope dilution gave similar estimates of nitrification rates. These rates were similar to those estimated by inhibition of ammonium oxidation in one soil but were lower in the other two soils. In the latter two soils, nitrification of labeled ammonium derived from dissimilatory nitrate reduction resulted in underestimation of nitrification rates by isotope dilution.     

Chloramphenicol inhibition of denitrifying enzyme activity in two agricultural soils.

Chloramphenicol, at concentrations greater than 0.1 g/liter (0.3 mM), inhibited the denitrifying enzyme activity (DEA) of slurries of humisol and sandy loam soils by disrupting the activity of existing nitrate reductase enzymes. When the concentration of chloramphenicol was increased from 0.1 to 2.0 g/liter (6.0 mM), the rate of nitrite production from nitrate decreased by 25 to 46%. The rate of NO production from nitrate decreased by 20 to 39%, and the rate of N(2)O production from nitrate, in the presence of acetylene (DEA), decreased by 21 to 61%. The predicted values of DEA at 0 g of chloramphenicol/liter computed from linear regressions of DEA versus chloramphenicol concentration were 18 to 43% lower than DEA measurements made in the absence of chloramphenicol and within a few per cent of DEA rates measured in the presence of 0.1 g of chloramphenicol/liter. We conclude that DEA assays should be carried out with a single (0.1-g/liter) chloramphenicol concentration. Chloramphenicol at concentrations greater than 0.1 g/liter inhibits the activity of existing denitrifying enzymes and should not be used in DEA assays.