Emerging complexity in the denitrification regulatory network of Bradyrhizobium japonicum

Bradyrhizobium japonicum is a Gram-negative soil bacterium symbiotically associated with soya bean plants, which is also able to denitrify under free-living and symbiotic conditions. In B. japonicum, the napEDABC, nirK, norCBQD and nosRZDYFLX genes which encode reductases for nitrate, nitrite, nitric oxide and nitrous oxide respectively are required for denitrification. Similar to many other denitrifiers, expression of denitrification genes in B. japonicum requires both oxygen limitation and the presence of nitrate or a derived nitrogen oxide. In B. japonicum, a sophisticated regulatory network consisting of two linked regulatory cascades co-ordinates the expression of genes required for microaerobic respiration (the FixLJ/FixK2 cascade) and for nitrogen fixation (the RegSR/NifA cascade). The involvement of the FixLJ/FixK2 regulatory cascade in the microaerobic induction of the denitrification genes is well established. In addition, the FNR (fumarase and nitrate reduction regulator)/CRP(cAMP receptor protein)-type regulator NnrR expands the FixLJ/FixK2 regulatory cascade by an additional control level. A role for NifA is suggested in this process by recent experiments which have shown that it is required for full expression of denitrification genes in B. japonicum. The present review summarizes the current understanding of the regulatory network of denitrification in B. japonicum.     

Regulation of nif gene expression in Enterobacter agglomerans: nucleotide sequence of the nifLA operon and influence of temperature and ammonium on its transcription

The nucleotide sequence of a plasmid-borne 3.9 kb XhoI-SmaI fragment comprising the 3′-region of the nifM gene, the nifL and nifA genes and the 5′-region of nifB gene of Enterobacter agglomerans was determined. The genes were identified by their homology to the corresponding nif genes of Klebsiella pneumoniae. A typical σ⁵⁴-dependent promoter and a consensus NtrC-binding motif were identified upstream of nifL. The predicted amino acid sequence of NifL showed close similarities to NifL of K. pneumoniae and Azotobacter vinelandii. However, no histidine residue was found to correspond to histidine-304 of A. vinelandii NifL, which had been proposed to be required for the repressor activity of NifL. The NifA sequence with a putative DNA binding motif (Q(X₃) A (X₃) G (X₅)I) and an ATP binding site in the C-terminal and central domains, respectively, resembles that of other known NifA proteins. The function of the nifL and nifA genes was demonstrated in vivo using a binary plasmid system by their ability to activate a nifH promoter-lacZ fusion at different temperatures and concentrations of NH ₄ ⁺ . Maximal promoter activity occurred at 25°C, and it appears that the sensitivity of NifA to elevated temperatures is independent of NifL. The expression of nifL inhibited promoter activity in the presence of NifA when the initial NH ₄ ⁺ concentration in the medium exceeded 4 mM.     

Functional difference between <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> NifA and <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> NifA

The nifA gene is an important regulatory gene and its product, NifA protein, regulates the expression of many nif genes involved in the nitrogen fixation process. We introduced multiple copies of the constitutively expressed Sinorhizobium meliloti (Sm) or Enterobacter cloacae (Ec) nifA gene into both the nifA mutant strain SmY and the wild-type strain Sm1021. Root nodules produced by SmY containing a constitutively expressed Sm nifA gene were capable of fixing nitrogen, while nodules produced by SmY containing the Ec nifA gene remained unable to fix nitrogen, as is the case for SmY itself. However, transfer of an additional Sm nifA gene into Sm1021 improved the nitrogen-fixing efficiency of root nodules to a greater extent than that observed upon transfer of the Ec nifA gene into Sm1021. Comparative analysis of amino acid sequences between Sm NifA and Ec NifA showed that the N-terminal domain was the least similar, but this domain is indispensable for complementation of the Fix^- phenotype of SmY by Sm NifA. We conclude that more than one domain is involved in determining functional differences between Sm NifA and Ec NifA.     

The regulatory status of the fixL- and fixJ-like genes in Bradyrhizobium japonicum may be different from that in Rhizobium meliloti

Summary The cloning, sequencing and mutational analysis of the Bradyrhizobium japonicum symbiotic nitrogen fixation genes fixL and fixJ are reported here. The two genes were adjacent and probably formed an operon, fixLJ. The predicted FixL and FixJ proteins, members of the two-component sensor/regulator family, were homologous over almost their entire lengths to the corresponding Rhizobium meliloti proteins (approx. 50% identity). Downstream of the B. japonicum fixJ gene was found an open reading frame with 138 codons (ORF138) whose product shared 36% homology with the N-terminal part of FixJ. Deletion and insertion mutations within fixL and fixJ led to a loss of approximately 90% wildtype symbiotic nitrogen fixation (Fix) activity, whereas an ORF138 mutant was Fix⁺. In fixL, fixJ and ORF138 mutant backgrounds, the aerobic expression of the fixR-nifA operon was not affected. NifA itself did not regulate the expression of the fixJ gene. Thus, the B. japonicum FixL and FixJ proteins were neither involved in the regulation of aerobic nifA gene expression nor in the anaerobic NifA-dependent autoregulation of the fixRnifA operon; rather they appeared to control symbiotically important genes other than those whose expression was dependent on the NifA protein. The fixL and fixJ mutant strains were unable to grow anaerobically with nitrate as the terminal electron acceptor. Therefore, some of the FixJ-dependent genes in B. japonicum may be concerned with anaerobic respiration.     

Denitrification activity of Bradyrhizobium sp. isolated from Argentine soybean cultivated soils

Two hundred and fifty strains, all of them representatives of native Bradyrhizobium sp., isolated from soils cultivated with soybean have been characterized by their denitrification activity. In addition, the denitrification potential of those soils was also measured by evaluating the most-probable-number (MPN) of denitrifying bacteria and the denitrification enzyme assay (DEA). Of the 250 isolates tested, 73 were scored as probable denitrifiers by a preliminary screening method. Only 41 were considered denitrifiers because they produced gas bubbles in Durham tubes, cultures reached an absorbance of more than 0.1 and NO³⁻ and NO²⁻ were not present. Ten of these 41 were selected to confirm denitrification and to study denitrification genes. According to N₂O production and cell protein concentration with NO³⁻, the isolates could be differentiated in three categories of denitrifiers. The presence of the napA, nirK, norC and nosZ genes was detected by production of a diagnostic PCR product using specific primers. RFLP from the 16S-23S rDNA spacer region (IGS) revealed that denitrifiers strains could be characterized as Bradyrhizobium japonicum and strains which were non-respiratory denitrifiers as B. elkanii.     

Ammonia oxidation and nitrate reduction marker genes are key indicators of nitrogen losses in temperate forest catchments

Chronic nitrogen inputs can alleviate N limitation and potentially impose N losses in forests, indicated by soil enrichment in ¹⁵N over ¹⁴N. However, the complexity of the nitrogen cycle hinders accurate quantification of N fluxes. Simultaneously, soil ecologists are striving to find meaningful indicators to characterise the “openness” of the nitrogen cycle. We integrate soil δ¹⁵N with constrained ecosystem N losses and the functional gene potential of the soil microbiome in 14 temperate forest catchments. We show that N losses are associated with soil δ¹⁵N and that δ¹⁵N scales with the abundance of soil bacteria. The abundance of the archaeal amoA gene, representing the first step in nitrification (ammonia oxidation to nitrite), followed by the abundance of narG and napA genes, associated with the first step in denitrification (nitrate reduction to nitrite), explains most of the variability in soil δ¹⁵N. These genes are more informative than the denitrification genes nirS and nirK, which are directly linked to N₂O production. Nitrite formation thus appears to be the critical step associated with N losses. Furthermore, we show that the genetic potential for ammonia oxidation and nitrate reduction is representative of forest soil ¹⁵N enrichment and thus indicative of ecosystem N losses.     

Regulation of nif gene expression in Enterobacter agglomerans: nucleotide sequence of the nifLA operon and influence of temperature and ammonium on its transcription

The nucleotide sequence of a plasmid-borne 3.9 kb XhoI-SmaI fragment comprising the 3-region of the nifM gene, the nifL and nifA genes and the 5-region of nifB gene of Enterobacter agglomerans was determined. The genes were identified by their homology to the corresponding nif genes of Klebsiella pneumoniae. A typical ⁵⁴-dependent promoter and a consensus NtrC-binding motif were identified upstream of nifL. The predicted amino acid sequence of NifL showed close similarities to NifL of K. pneumoniae and Azotobacter vinelandii. However, no histidine residue was found to correspond to histidine-304 of A. vinelandii NifL, which had been proposed to be required for the repressor activity of NifL. The NifA sequence with a putative DNA binding motif (Q(X₃) A (X₃) G (X₅)I) and an ATP binding site in the C-terminal and central domains, respectively, resembles that of other known NifA proteins. The function of the nifL and nifA genes was demonstrated in vivo using a binary plasmid system by their ability to activate a nifH promoter-lacZ fusion at different temperatures and concentrations of NH ₄ ⁺ . Maximal promoter activity occurred at 25°C, and it appears that the sensitivity of NifA to elevated temperatures is independent of NifL. The expression of nifL inhibited promoter activity in the presence of NifA when the initial NH ₄ ⁺ concentration in the medium exceeded 4 mM.Communicated by H. Böhme     

The Impact of Using Mature Compost on Nitrous Oxide Emission and the Denitrifier Community in the Cattle Manure Composting Process

The diversity and dynamics of the denitrifying genes (nirS, nirK, and nosZ) encoding nitrite reductase and nitrous oxide (N₂O) reductase in the dairy cattle manure composting process were investigated. A mixture of dried grass with a cattle manure compost pile and a mature compost-added pile were used, and denaturing gradient gel electrophoresis was used for denitrifier community analysis. The diversity of nirK and nosZ genes significantly changed in the initial stage of composting. These variations might have been induced by the high temperature. The diversity of nirK was constant after the initial variation. On the other hand, the diversity of nosZ changed in the latter half of the process, a change which might have been induced by the accumulation of nitrate and nitrite. The nirS gene fragments could not be detected. The use of mature compost that contains nitrate and nitrite promoted the N₂O emission and significantly affected the variation of nosZ diversity in the initial stage of composting, but did not affect the variation of nirK diversity. Many Pseudomonas-like nirK and nosZ gene fragments were detected in the stage in which N₂O was actively emitted.     

不同土地利用方式土壤氨氧化微生物和反硝化微生物时空分布特征

研究不同土地利用方式下氮循环相关微生物在不同土壤剖面的分布,可为认识和理解土壤氮转化过程提供科学依据。土壤氨氧化微生物和反硝化微生物在调节氮肥利用率、硝态氮淋溶和氧化亚氮(N₂O)排放等方面有着重要作用。以北京郊区农田和林地两种土地利用方式为研究对象,分析土壤氨氧化潜势和亚硝酸盐氧化潜势在0—100 cm土壤剖面上的季节分布(春季和秋季),并通过实时荧光定量PCR方法表征土壤氨氧化和反硝化微生物的时空分布特征。结果表明,农田土壤氨氧化潜势、亚硝酸盐氧化潜势、氨氧化微生物和反硝化微生物丰度均显著高于林地土壤,且随土壤深度增加而显著降低。除氨氧化古菌amoA基因丰度在不同季节间无显著差异外,春季土壤氨氧化细菌(amoA基因)、反硝化微生物nirS、nirK和典型nosZ I基因的丰度均显著高于秋季。土壤有机质、总氮、NH~+₄-N、NO~-₃-N含量与氨氧化微生物和反硝化微生物的功能基因丰度显著相关。综上,不同土地利用方式下土壤氮循环相关微生物的丰度与土壤氮素的可利用性和转化过程紧密相关,研究结果对土壤氮素利用和养分管理提供...     

Isolation,genetic and functional characterization of novel soil nirK-type denitrifiers

Denitrification, the reduction of nitrogen oxides (NO₃⁻ and NO₂⁻) to N₂ via the intermediates NO and N₂O, 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.     

Diversity,abundance, and distribution of NO‐forming nitrite reductase–encoding genes in deep‐sea subsurface sediments of the South China Sea

In marine ecosystems, both nitrite‐reducing bacteria and anaerobic ammonium‐oxidizing (anammox) bacteria, containing different types of NO‐forming nitrite reductase–encoding genes, contribute to the nitrogen cycle. The objectives of study were to reveal the diversity, abundance, and distribution of NO‐forming nitrite reductase–encoding genes in deep‐sea subsurface environments. Results showed that higher diversity and abundance of nirS gene than nirK and Scalindua‐nirS genes were evident in the sediments of the South China Sea (SCS), indicating bacteria containing nirS gene dominated the NO‐forming nitrite‐reducing microbial community in this ecosystem. Similar diversity and abundance distribution patterns of both nirS and Scalindua‐nirS genes were detected in this study sites, but different from nirK gene. Further statistical analyses also showed both nirS and Scalindua‐nirS genes respond similarly to environmental factors, but differed from nirK gene. These results suggest that bacteria containing nirS and Scalindua‐nirS genes share similar niche in deep‐sea subsurface sediments of the SCS, but differed from those containing nirK gene, indicating that community structures of nitrite‐reducing bacteria are segregated by the functional modules (NirS vs. NirK) rather than the competing processes (anammox vs. classical denitrification).     

氮磷添加对草甸草原土壤氮磷转化功能基因丰度的影响

氮添加是提高退化草地生产力的主要养分管理措施,而过量的氮输入会导致土壤酸化、增加硝酸盐淋溶损失和温室气体排放。旨在明确草原割草利用下土壤氮、磷转化功能基因丰度对氮磷添加的响应规律,为定向调控打草场土壤氮、磷转化过程,提高养分利用效率,减少温室气体N₂O排放提供科学依据。2018—2020年在呼伦贝尔草甸草原打草场设置了5个施氮水平(0、1.55、4.65、13.95、27.9 g N m^-2 a^-1)和3个磷水平(0、5.24、10.48 g P m^-2 a^-1),裂区试验设计,在植物不同生长时期测定土壤氨氧化(amoA-AOA和amoA-AOB)、反硝化(narG、nirK、nirS和nosZ)和磷转化(phoD)基因丰度。结果表明,土壤氮转化基因丰度受到氮、磷添加的调控,而氮、磷添加对土壤磷转化功能基因丰度无显著影响(P>0.05)。氮添加可提高amoA-AOB基因丰度,增加氨氧化细菌调控土壤总硝化速率的相对重要性,因此能增加硝酸盐淋溶损失潜势。高氮处理下添加磷可降低...     

Genetic and Environmental Controls on Nitrous Oxide Accumulation in Lakes

We studied potential links between environmental factors, nitrous oxide (N₂O) accumulation, and genetic indicators of nitrite and N₂O reducing bacteria in 12 boreal lakes. Denitrifying bacteria were investigated by quantifying genes encoding nitrite and N₂O reductases (nirS/nirK and nosZ, respectively, including the two phylogenetically distinct clades nosZ _I and nosZ _II) in lake sediments. Summertime N₂O accumulation and hypolimnetic nitrate concentrations were positively correlated both at the inter-lake scale and within a depth transect of an individual lake (Lake Vanajavesi). The variability in the individual nirS, nirK, nosZ _I, and nosZ _II gene abundances was high (up to tenfold) among the lakes, which allowed us to study the expected links between the ecosystem’s nir-vs-nos gene inventories and N₂O accumulation. Inter-lake variation in N₂O accumulation was indeed connected to the relative abundance of nitrite versus N₂O reductase genes, i.e. the (nirS+nirK)/nosZ _I gene ratio. In addition, the ratios of (nirS+nirK)/nosZ _I at the inter-lake scale and (nirS+nirK)/nosZ _I+II within Lake Vanajavesi correlated positively with nitrate availability. The results suggest that ambient nitrate concentration can be an important modulator of the N₂O accumulation in lake ecosystems, either directly by increasing the overall rate of denitrification or indirectly by controlling the balance of nitrite versus N₂O reductase carrying organisms.     

Intracellular nitrite accumulation: The cause of growth inhibition of Microcystis aeruginosa exposure to high nitrite level

The aim of the present study is to test the role of intracellular nitrite in external nitrite suppressing algal growth. We examined the growth of Microcystis aeruginosa at different nitrite levels under high nitrate conditions and without nitrate conditions. There were higher intracellular nitrite and lower P_m^chla, R_d ^chla, α^chl, maximum cell density and specific growth rate in high nitrate group than nitrate absence group at 5 mg NO₂^?‐N L^?1. At 10 and 15 mg NO₂^?‐N L^?1, P_m^chla, R_d ^chla, α^chl, maximum cell densities and specific growth rates in the high nitrate group became higher than those of the nitrate absence group, while a lower intracellular nitrite in the high nitrate group than nitrate absence group was observed. In addition, the intracellular nitrite and the growth of M. aeruginosa in the high nitrate group did not change from 5 to 10 mg NO₂^?‐N L^?1. In the nitrite uptake experiment, with nitrite concentration increasing from 5 to 15 mg NO₂^?‐N L^?1, maximum nitrite uptake rate of alga increased, and half‐saturation constant of alga decreased. These results indicate that external nitrite inhibited algal growth through stimulating intracellular nitrite rise, which resulted from overexpression of nitrite transporter.     

Abundance of narG, nirS, nirK, and nosZ Genes of Denitrifying Bacteria during Primary Successions of a Glacier Foreland

Quantitative PCR of denitrification genes encoding the nitrate, nitrite, and nitrous oxide reductases was used to study denitrifiers across a glacier foreland. Environmental samples collected at different distances from a receding glacier contained amounts of 16S rRNA target molecules ranging from 4.9 × 10⁵ to 8.9 × 10⁵ copies per nanogram of DNA but smaller amounts of narG, nirK, and nosZ target molecules. Thus, numbers of narG, nirK, nirS, and nosZ copies per nanogram of DNA ranged from 2.1 × 10³ to 2.6 × 10⁴, 7.4 × 10² to 1.4 × 10³, 2.5 × 10² to 6.4 × 10³, and 1.2 × 10³ to 5.5 × 10³, respectively. The densities of 16S rRNA genes per gram of soil increased with progressing soil development. The densities as well as relative abundances of different denitrification genes provide evidence that different denitrifier communities develop under primary succession: higher percentages of narG and nirS versus 16S rRNA genes were observed in the early stage of primary succession, while the percentages of nirK and nosZ genes showed no significant increase or decrease with soil age. Statistical analyses revealed that the amount of organic substances was the most important factor in the abundance of eubacteria as well as of nirK and nosZ communities, and copy numbers of these two genes were the most important drivers changing the denitrifying community along the chronosequence. This study yields an initial insight into the ecology of bacteria carrying genes for the denitrification pathway in a newly developing alpine environment.     

In Bradyrhizobium japonicum the common nodulation genes, nodABC, are linked to nifA and fixA

Summary Using cloned Rhizobium phaseoli nodulation (nod) genes as hybridization probes homologous restriction fragments were detected in the genome of the slow-growing soybean symbiont, Bradyrhizobium japonicum strain 110. These fragments were isolated from a cosmid library, and were shown to lie 10 kilobasepairs (kb) upstream from the nifA and fixA genes. Specific nod probes from Rhizobium leguminosarum were used to identify nodA-, nodB-, and nodC-like sequences clustered within a 4.5 kb PstI fragment. A mutant was constructed in which the kanamycin resistance gene from Tn5 was inserted into the nodA homologous B. japonicum region. This insertion was precisely located, by DNA sequencing, to near the middle of the nodA gene. B. japonicum mutants carrying this insertion were completely nodulation deficient (Nod^-).