nifH Copies Research Articles

Seasonal changes in total mercury and methylmercury in subtropical decomposing litter correspond to the abundances of nitrogen-fixing and methylmercury-degrading bacteria.

Previous research has found total mercury (THg) and methylmercury (MeHg) levels increase with litterfall decay, thus suggesting litterfall decomposition plays an essential role in the biogeochemical transformation of mercury (Hg). However, it remains unclear how Hg accumulates in the decaying litter, how bacterial taxa networks vary and what roles various microorganisms play during litterfall decomposition, especially nitrogen (N)-fixing, MeHg-degrading and Hg-methylating microbes. Here, we demonstrated as degradation proceeded, a gradually-complex network evolved for litterfall bacteria for the subtropical mixed broadleaf-conifer (MBC) forest, whereas a relatively static network existed for the evergreen broadleaf (EB) forest. N-fixing and MeHg-degrading bacteria dominated throughout litterfall decomposition process, with relative abundances of N-fixing genera and nifH copies maximum and relative abundances of MeHg-degrading bacteria and merAB copies minimum in summer. Hence, N-fixing bacteria likely mediate THg increase in the decomposing litterfall, while MeHg enhancement may be regulated by aerobic MeHg-degrading microbes which can transform MeHg to inorganic divalent Hg (Hg2+) or further to elemental Hg (Hg0). Together, this work elucidates variations of N-fixing and MeHg-degrading microbes in decaying litterfall and their relationships with Hg accumulation, providing novel insights into understanding the biogeochemical cycle of Hg in the forest ecosystem.

Cropland‐to‐Miscanthus conversion alters soil bacterial and archaeal communities influencing N‐cycle in Northern China

AbstractMiscanthus spp. are increasingly cultivated in cropland worldwide due to their bioenergy potential and multiple ecological services. Effects of long‐term cropland‐to‐Miscanthus conversion without N fertilizer on soil microbiome and N cycling largely remain unknown. We aimed to explore the effects of Miscanthus conversion on soil microbiome and N cycling over a 15‐year period. We analyzed diversity, composition, and abundance of bacterial and archaeal communities using 16S rRNA amplicon sequencing, and abundances of N‐cycling‐related genes using quantitative polymerase chain reaction of 0–10 cm soils collected from bare land, cropland, 10‐year Miscanthus × giganteus, and 15‐year Miscanthus sacchriflorus land in Beijing. Conversion decreased soil sand and micro‐aggregate proportion, nitrate N (NiN), available phosphorus levels, conductivity, temperature, and pH, while increasing proportion of soil clay and macro‐aggregate (MAA), soil organic C (SOC), available N (AN), exchangeable Mg2+ (EMg2+), and available potassium (AK) contents as well as microbial C/N. Consequently, diversity, composition, and abundance of soil bacterial community exhibited larger changes than those values of archaeal community after conversion. Soil AP, EMg2+, AK, and SOC were key factors in shifting microbiome from the cropland to Miscanthus pattern. Moreover, abundances of bacterial and archaeal communities and the N fixer gene nifH increased, whereas that of the bacterial ammonia monooxygenase gene decreased. The copies of other N‐cycling‐related genes in the two Miscanthus lands seemed similar to those values of cropland. The nifH copies negatively correlated with soil NiN and positively correlated with AN, EMg2+, ECa2+, SOC, AK, and MAA. We conclude that changes in soil microbiome pattern induced by the variation of soil properties enhance microbial N fixation potential, maintaining stable N levels and robust N cycling with lower N leakage risk after conversion. These results should inspire farmers and governments to large‐scale use Miscanthus on marginal cropland in Northern China.

Programmed cell death in diazotrophs and the fate of organic matter in the western tropical South Pacific Ocean during the OUTPACE cruise

Abstract. The fate of diazotroph (N2 fixers) derived carbon (C) and nitrogen (N) and their contribution to vertical export of C and N in the western tropical South Pacific Ocean was studied during OUTPACE (Oligotrophy to UlTra-oligotrophy PACific Experiment). Our specific objective during OUTPACE was to determine whether autocatalytic programmed cell death (PCD), occurring in some diazotrophs, is an important mechanism affecting diazotroph mortality and a factor regulating the vertical flux of organic matter and, thus, the fate of the blooms. We sampled at three long duration (LD) stations of 5 days each (LDA, LDB and LDC) where drifting sediment traps were deployed at 150, 325 and 500 m depth. LDA and LDB were characterized by high chlorophyll a (Chl a) concentrations (0.2–0.6 µg L−1) and dominated by dense biomass of the filamentous cyanobacterium Trichodesmium as well as UCYN-B and diatom–diazotroph associations (Rhizosolenia with Richelia-detected by microscopy and het-1 nifH copies). Station LDC was located at an ultra-oligotrophic area of the South Pacific gyre with extremely low Chl a concentration (∼ 0.02 µg L−1) with limited biomass of diazotrophs predominantly the unicellular UCYN-B. Our measurements of biomass from LDA and LDB yielded high activities of caspase-like and metacaspase proteases that are indicative of PCD in Trichodesmium and other phytoplankton. Metacaspase activity, reported here for the first time from oceanic populations, was highest at the surface of both LDA and LDB, where we also obtained high concentrations of transparent exopolymeric particles (TEP). TEP were negatively correlated with dissolved inorganic phosphorus and positively coupled to both the dissolved and particulate organic carbon pools. Our results reflect the increase in TEP production under nutrient stress and its role as a source of sticky carbon facilitating aggregation and rapid vertical sinking. Evidence for bloom decline was observed at both LDA and LDB. However, the physiological status and rates of decline of the blooms differed between the stations, influencing the amount of accumulated diazotrophic organic matter and mass flux observed in the traps during our experimental time frame. At LDA sediment traps contained the greatest export of particulate matter and significant numbers of both intact and decaying Trichodesmium, UCYN-B and het-1 compared to LDB where the bloom decline began only 2 days prior to leaving the station and to LDC where no evidence for bloom or bloom decline was seen. Substantiating previous findings from laboratory cultures linking PCD to carbon export in Trichodesmium, our results from OUTPACE indicate that nutrient limitation may induce PCD in high biomass blooms such as displayed by Trichodesmium or diatom–diazotroph associations. Furthermore, PCD combined with high TEP production will tend to facilitate cellular aggregation and bloom termination and will expedite vertical flux to depth.

Methanotrophs are core members of the diazotroph community in decaying Norway spruce logs

Dead wood is initially a nitrogen (N) poor substrate, where the N content increases with decay, partly due to biological N2 fixation, but the drivers of the N accumulation are poorly known. We quantified the rate of N2 fixation in decaying Norway spruce logs of different decay stages and studied the potential regulators of the N2-fixation activity. The average rate for acetylene reduction in the decaying wood was 7.5 nmol ethylene g−1d−1, which corresponds to 52.9 μg N kg−1d−1. The number of nifH copies (g−1 dry matter) was higher at the later decay stages, but no correlation between the copy number and the in vitro N2 fixation rate was found. All recovered nifH sequences were assigned to the order Rhizobiales, and therein mostly (60%) to methane oxidizing genera. We confirm that nitrogen fixing methanotrophs are present in all the wood decay phases and suggest that their interaction between methane producing organisms in decaying wood should be further studied.

Temporal Variability of Trichodesmium spp. and Diatom-Diazotroph Assemblages in the North Pacific Subtropical Gyre

In oligotrophic ocean regions such as the North Pacific Subtropical Gyre (NPSG), N2 fixation (i.e. diazotrophy) by a diverse consortium of microorganisms has been shown to contribute significantly to new production and particle export. In 2015 and 2016, we measured near-monthly abundances of the large cell-sized (> 10 m) diazotrophic genera Trichodesmium and diatom-associated Richelia and Calothrix spp. in the NPSG via microscopy and quantitative PCR of nifH genes. Of these genera, we find Trichodesmium to be the more abundant over our study period, with cell concentrations in the upper water column (0-45 m) ranging from 1-5988 cells L-1, while the sum of Richelia and Calothrix spp. abundances ranged from 4-157 heterocysts L-1. Significant discrepancies between absolute abundances were noted between cell and gene-based approaches to biomass determination (nifH copies L-1 were up to 102-103 higher than cell concentrations). Potential explanations for these striking discrepancies are discussed. Using the maximum N2 fixation rates per cell found in the existing literature for these genera, we estimate potential N2 fixation rates via these large diazotroph communities to be between 0.01-1.5 nmol N L-1 d-1. When comparing these rates to available 15N2 tracer measurements, we conclude that large diazotrophs were generally minor (50% of measured N2 fixation rates. While these large cell-sized and heterogeneously distributed organisms may still disproportionately contribute to export, cell-abundance based rate estimates suggests that other diazotrophs are largely responsible for N2 fixation rates measured in bottle-based incubations.

Biological N2 Fixation in the Upwelling Region off NW Iberia: Magnitude, Relevance, and Players

The classical paradigm about marine N2 fixation establishes that this process is mainly constrained to nitrogen-poor tropical and subtropical regions, and sustained by the colonial cyanobacterium Trichodesmium spp. and diatom-diazotroph symbiosis. However, the application of molecular techniques allowed determining a high phylogenic diversity and a wide distribution of marine diazotrophs, which extends the range of ocean environments where biological N2 fixation may be relevant. Between February 2014 and December 2015, we carried out 10 one-day samplings in the upwelling system off NW Iberia in order to: 1) investigate the seasonal variability in the magnitude of N2 fixation, 2) determine its biogeochemical role as a mechanism of new nitrogen supply, and 3) quantify the main diazotrophs in the region under contrasting hydrographic regimes. Our results indicate that the magnitude of N2 fixation in this region was relatively low (0.001±0.002 – 0.095±0.024 µmol N m-3 d-1), comparable to the lower-end of rates described for the subtropical NE Atlantic. Maximum rates were observed at surface during both upwelling and relaxation conditions. The comparison with nitrate diffusive fluxes revealed the minor role of N2 fixation (2 fixation activity detected in the region. Quantitative PCR targeting the nifH gene revealed the highest abundances of two sublineages of Candidatus Atelocyanobacterium thalassa or UCYN-A (UCYN-A1 and UCYN-A2) mainly at surface waters during upwelling and relaxation conditions, and of Gammaproteobacteria γ-24774A11 at deep waters during downwelling. Maximum abundance for the three groups were up to 6.7 × 102, 1.5 × 103 and 2.4 × 104 nifH copies L-1, respectively. Our findings demonstrate measurable N2 fixation activity and presence of diazotrophs throughout the year in a nitrogen-rich temperate region.

Mesozooplankton Graze on Cyanobacteria in the Amazon River Plume and Western Tropical North Atlantic.

Diazotrophic cyanobacteria, those capable of fixing di-nitrogen (N2), are considered one of the major sources of new nitrogen (N) in the oligotrophic tropical ocean, but direct incorporation of diazotrophic N into food webs has not been fully examined. In the Amazon River-influenced western tropical North Atlantic (WTNA), diatom diazotroph associations (DDAs) and the filamentous colonial diazotrophs Trichodesmium have seasonally high abundances. We sampled epipelagic mesozooplankton in the Amazon River plume and WTNA in May–June 2010 to investigate direct grazing by mesozooplankton on two DDA populations: Richelia associated with Rhizosolenia diatoms (het-1) and Hemiaulus diatoms (het-2), and on Trichodesmium using highly specific qPCR assays targeting nitrogenase genes (nifH). Both DDAs and Trichodesmium occurred in zooplankton gut contents, with higher detection of het-2 predominantly in calanoid copepods (2.33–16.76 nifH copies organism-1). Abundance of Trichodesmium was low (2.21–4.03 nifH copies organism-1), but they were consistently detected at high salinity stations (>35) in calanoid copepods. This suggests direct grazing on DDAs, Trichodesmium filaments and colonies, or consumption as part of sinking aggregates, is common. In parallel with the qPCR approach, a next generation sequencing analysis of 16S rRNA genes identified that cyanobacterial assemblage associated with zooplankton guts was dominated by the non-diazotrophic unicellular phylotypes Synechococcus (56%) and Prochlorococcus (26%). However, in two separate calanoid copepod samples, two unicellular diazotrophs Candidatus Atelocyanobacterium thalassa (UCYN-A) and Crocosphaera watsonii (UCYN-B) were present, respectively, as a small component of cyanobacterial assemblages (<2%). This study represents the first evidence of consumption of DDAs, Trichodesmium, and unicellular cyanobacteria by calanoid copepods in an area of the WTNA known for high carbon export. These diazotroph populations are quantitatively important in the global N budget, widespread and hence, the next step is to accurately quantify grazing. Nonetheless, these results highlight a direct pathway of diazotrophic N into the food web and have important implications for biogeochemical cycles, particularly oligotrophic regions where N2 fixation is the main source of new nitrogen.

Contrasting bacterial communities in two indigenous Chionochloa (Poaceae) grassland soils in New Zealand.

The cultivation of grasslands can modify both bacterial community structure and impact on nutrient cycling as well as the productivity and diversity of plant communities. In this study, two pristine New Zealand grassland sites dominated by indigenous tall tussocks (Chionochloa pallens or C. teretifolia) were examined to investigate the extent and predictability of variation of the bacterial community. The contribution of free-living bacteria to biological nitrogen fixation is predicted to be ecologically significant in these soils; therefore, the diazotrophic community was also examined. The C. teretifolia site had N-poor and poorly-drained peaty soils, and the C. pallens had N-rich and well-drained fertile soils. These soils also differ in the proportion of organic carbon (C), Olsen phosphorus (P) and soil pH. The nutrient-rich soils showed increased relative abundances of some copiotrophic bacterial taxa (including members of the Proteobacteria, Bacteroidetes and Firmicutes phyla). Other copiotrophs, Actinobacteria and the oliogotrophic Acidobacteria showed increased relative abundance in nutrient-poor soils. Greater diversity based on 16S rRNA gene sequences and the Tax4Fun prediction of enhanced spore formation associated with nutrient-rich soils could indicate increased resilience of the bacterial community. The two sites had distinct diazotrophic communities with higher diversity in C. teretifolia soils that had less available nitrate and ammonium, potentially indicating increased resilience of the diazotroph community at this site. The C. teretifolia soils had more 16S rRNA gene and nifH copies per g soil than the nutrient rich site. However, the proportion of the bacterial community that was diazotrophic was similar in the two soils. We suggest that edaphic and vegetation factors are contributing to major differences in the composition and diversity of total bacterial and diazotrophic communities at these sites. We predict the differences in the communities at the two sites will result in different responses to environmental change.

Longitudinal variability of diazotroph abundances in the subtropical North Atlantic Ocean

Diazotrophy-related studies in the North Atlantic have largely focused on its western tropical area, leaving the subtropics and the east undersampled. We studied the longitudinal distribution of Trichodesmium, UCYN-A, UCYN-B, the putative Gammaproteobacterium g-24774A11 and Richelia (Het1) along 24.5 degrees N, using quantitative polymerase chain reaction on different size fractions (10, 10-3 and 3-0.2 mm) and additional filament counts for Trichodesmium. Trichodesmium was the most abundant phylotype, followed by UCYN-A, gamma-24774A11 and Het1, with maximum abundances of 8.8 x 10(5), 2.0 x 10(5), 3.3 x 10(3) and 3.4 x 10(2) nifH copies L-1, respectively, whereas UCYN-B was mostly undetected. A clear shift in the diazotroph community was observed at similar to 30 degrees W, coinciding with the transition between the North Atlantic Subtropical Gyre boundary and inner core. This transition zone divided the transect into an eastern half dominated by UCYN-A and western half dominated by Trichodesmium and gamma-24774A11. gamma-24774A11 was only detected in the 10-3 mu m fraction, suggesting their association with larger microbes or aggregates. Our results indicate that typical size fractionation by 10 mu m is not optimal for reconciling diazotroph phylotypes to N-2 fixation rates and that non-cyanobacterial diazotrophs may contribute importantly to bulk diazotrophic activity in the western subtropical North Atlantic.

Isolation of heterotrophic diazotrophic bacteria from estuarine surface waters

The wide distribution of diverse nitrogenase (nifH) genes affiliated with those of heterotrophic bacteria in marine and estuarine waters indicates ubiquity and an ecologically relevant role for heterotrophic N2 -fixers (diazotrophs) in aquatic nitrogen (N) cycling. However, the lack of cultivated representatives currently precludes an evaluation of their N2 -fixing capacity. In this study, microoxic or anoxic N-free media were inoculated with estuarine Baltic Sea surface water to select for N2 -fixers. After visible growth and isolation of single colonies on oxic plates or in anoxic agar tubes, nifH gene amplicons were obtained from 64 strains and nitrogenase activity, applying the acetylene reduction assay, was confirmed for 40 strains. Two strains, one Gammaproteobacterium affiliated with Pseudomonas and one Alphaproteobacterium affiliated with Rhodopseudomonas were shown to represent established members of the indigenous diazotrophic community in the Baltic Sea, with abundances of up to 7.9 × 10(4) and 4.7 × 10(4) nifH copies l(-1) respectively. This study reports media for successful isolation of heterotrophic diazotrophs. The applied methodology and the obtained strains will facilitate future identification of factors controlling heterotrophic diazotrophic activity in aquatic environments, which is a prerequisite for understanding and evaluating their ecology and contribution to N cycling at local and regional scales.

Diazotroph community structure and abundance in wheat–fallow and wheat–pea crop rotations

Biological input of nitrogen (N) from the atmosphere by free-living diazotrophs can help alleviate fertilizer use in agricultural systems. In this study, we investigated the effect of N fertilizer and winter pea (Pisum sativum L.) crop on the community structure and abundance of free-living diazotrophs in a two year study of dryland winter wheat (Triticum aestivum L.) no-till production system in Eastern Oregon, USA. Based on quantification of the nifH gene, diazotroph abundance was strongly influenced by plant species and the crop year in which the soil samples were collected. A greater amount of nifH copies was recovered in 2012 compared to 2011 either as copies per gram soil or normalized to the abundance of bacterial 16S rRNA genes. The quantity of genes was greater under pea than wheat in 2012 although no difference was observed in the preceding year. The nifH gene abundance was positively correlated to ammonium concentration in 2011 and bacterial abundance in 2012. Nitrogen application did not influence diazotroph abundance in the top 0–5 cm; however the abundance was reduced by application at the lower 5–10 cm depth under wheat crop. The diazotroph community structure appeared to be influenced more by N fertilization rather than plant species with the exception of wheat in 2012. Changes in the community structure over the two years were greater for fertilized than unfertilized soil. Collectively, these data suggest that year-to-year variability had a greater influence on diazotroph communities rather than specific parameters of plant species, fertilization, total N, total organic C, or soil pH. Multi-year studies are necessary to define the specific drivers of diazotroph abundance, community structure and function.

Active nitrogen-fixing heterotrophic bacteria at and below the chemocline of the central Baltic Sea

The Baltic Sea receives large nitrogen inputs by diazotrophic (N2-fixing) heterocystous cyanobacteria but the significance of heterotrophic N2 fixation has not been studied. Here, the diversity, abundance and transcription of the nifH fragment of the nitrogenase enzyme in two basins of the Baltic Sea proper was examined. N2 fixation was measured at the surface (5 m) and in anoxic water (200 m). Vertical sampling profiles of >10 and <10 μm size fractions were collected in 2007, 2008 and 2011 at the Gotland Deep and in 2011 in the Bornholm Basin. Both of these stations are characterized by permanently anoxic bottom water. The 454-pyrosequencing nifH analysis revealed a diverse assemblage of nifH genes related to alpha-, beta- and gammaproteobacteria (nifH cluster I) and anaerobic bacteria (nifH cluster III) at and below the chemocline. Abundances of genes and transcripts of seven diazotrophic phylotypes were investigated using quantitative polymerase chain reaction revealing abundances of heterotrophic nifH phylotypes of up to 2.1 × 10(7) nifH copies l(-1). Abundant nifH transcripts (up to 3.2 × 10(4) transcripts l(-1)) within nifH cluster III and co-occurring N2 fixation (0.44±0.26 nmol l(-1) day(-1)) in deep water suggests that heterotrophic diazotrophs are fixing N2 in anoxic ammonium-rich waters. Our results reveal that N2 fixation in the Baltic Sea is not limited to illuminated N-deplete surface waters and suggest that N2 fixation could also be of importance in other suboxic regions of the world's oceans.

Distribution and activity of diazotrophs in the Eastern Equatorial Atlantic

The gene abundance and gene expression of six diazotroph populations from the Eastern Equatorial Atlantic in June 2007 were examined using nifH gene quantitative polymerase chain reaction (q PCR) methods. Of all the diazotrophs, Trichodesmium spp. was the most abundant with the highest number of gene copies in the Gulf of Guinea. Trichodesmium also had the highest nitrogenase gene transcript abundance overall with the maximum in samples collected at the equator and in waters influenced by the Congo River plume (> 105 cDNA nifH copies l−1). Both cyanobacterial unicellular groups (A and B) were detected, where group A was the second most abundant in surface samples, in particular at the stations along the equator. Transcript abundance for group A, however, was at the detection limit and suggests that it was not actively fixing N2. Trichodesmium and group B nifH gene abundances co-varied (P < 0.0001). Richelia associated with Hemiaulus hauckii diatoms were detected in 9 of 10 surface samples and the highest abundances (> 104nifH copies l−1) were found north-west of the Congo River plume. In contrast, the Calothrix symbionts (het-3) of Chaetoceros had low abundances at the surface, but were present at 3.7 × 104nifH copies l−1 at 40 m depth in the equatorial upwelling. This is the first report of the Calothrix symbiont in the Atlantic Ocean. This is also the first report of nifH gene copy and transcript abundance in an Equatorial upwelling zone. Although the number of gene copies for Richelia associated with Rhizosolenia were the lowest, the transcript abundance were high (9.4 × 101−1.8 × 104 cDNA nifH copies l−1) and similar to that of Trichodesmium. The distribution of the diazotroph groups, especially the three strains of symbiotic cyanobacteria, was different, and appeared largely controlled by riverine inputs and upwelling.

Regional distributions of nitrogen‐fixing bacteria in the Pacific Ocean

We evaluated the regional distributions of six nitrogen (N2)‐fixing bacteria in the North Pacific Ocean using quantitative polymerase chain reaction amplification of planktonic nifH genes. Samples were collected on four oceanographic research cruises between March 2002 and May 2005 that spanned a latitudinal range from 12°S and 54°N between 152°W and 170°W. Samples were collected throughout the upper ocean (&lt;200 m) in the northern regions of the South Pacific Subtropical Gyre (SPSG), equatorial waters, the North Pacific Subtropical Gyre (NPSG), the North Pacific Transitional Zone (NPTZ), and within the Pacific Sub Arctic Gyre (PSAG). There were distinct spatial gradients in concentrations of nutrients, chlorophyll, and the abundances of N2‐fixing bacteria within the various oceanic biomes. In general, nifH‐containing bacteria were most abundant in the midregions of the NPSG (latitudes between ~14°N and 29°N), where unicellular cyanobacterial phylotypes dominated nifH gene abundances. The abundances of all nifH‐containing groups declined within the northern and southern regions of NPSG. Although nifH‐containing groups were detectable in the northern regions of the SPSG, throughout the equatorial waters, and within the NPTZ, gene copy abundances of most groups were lower in these regions than those found the in the NPSG. In the NPSG, surface water abundances of the various nifH phylotypes examined ranged from &lt;50 copies L−1 to ~105 nifH copies LS−1. Overall, the abundances of an uncultivated, presumed unicellular nifH sequence‐type (termed Group A) were the most abundant and widely distributed of the phylotypes examined. Our results indicate that the distributions of N2‐fixing plankton were largely restricted to the subtropical regions of the North and South Pacific Oceans.

Three distinct clades of cultured heterocystous cyanobacteria constitute the dominant N2-fixing members of biological soil crusts of the Colorado Plateau, USA

The identity of the numerically dominant N(2)-fixing bacteria in biological soil crusts of the Colorado Plateau region and two outlying areas was determined using multiple approaches, to link the environmental diversity of nifH gene sequences to cultured bacterial isolates from the regions. Of the nifH sequence-types detected in soil crusts of the Colorado Plateau, 89% (421/473) were most closely related to nifH signature sequences from cyanobacteria of the order Nostocales. N(2)-fixing cyanobacterial strains were cultured from crusts and their morphotypes, 16S rRNA gene and nifH gene sequences were characterized. The numerically dominant diazotrophs in the Colorado Plateau crusts fell within three clades of heterocystous cyanobacteria. Two clades are well-represented by phylogenetically and morphologically coherent strains, corresponding to the descriptions of Nostoc commune and Scytonema hyalinum, which are widely recognized as important N(2)-fixing components of soil crusts. A third, previously-overlooked clade was represented by a phylogenetically coherent but morphologically diverse group of strains that encompass the morphogenera Tolypothrix and Spirirestis. Many of the strains in each of these groups contained at least two nifH copies that represent different clusters in the nifH environmental survey.

Characterization of the Rhizobium leguminosarum biovar phaseoli nifA gene, a positive regulator of nif gene expression.

We report the isolation, mutational analysis and the nucleotide sequence of the Rhizobium leguminosarum bv. phaseoli nifA gene. Comparison of the deduced amino acid sequence with other NifA sequences indicated the presence of the conserved central activator and the C-terminal DNA-binding domains. Nodules elicited by a R. leguminosarum bv. phaseoli nifA mutant were symbiotically ineffective. The expression of a nifA-gusA fusion was shown to be independent on the oxygen status of the cell. We cloned the three nifH copies of R. leguminosarum bv. phaseoli and determined the nucleotide sequence of their promoter regions. The expression of nifH-gusA fusions is induced under microaerobic conditions and is dependent on the presence of NifA.

Nucleotide Sequence and Functional Analysis of the Two nifH Copies of Rhizobium ORS571

Summary: In Rhizobium ORS571, which is able to form both root and stem nodules on the tropical plant Sesbania rostrata and to grow in the free-living state at the expense of N2, two copies of the nifH gene, which codes for the nitrogenase Fe-protein, were characterized. One copy, nifH1, was localized in the nifHDK operon; the other, nifH2, was localized elsewhere. nifH1 and nifH2 differed in only six nucleotides and both encoded a polypeptide of 296 amino acids with a single change at position 282, a serine in nifH1 and a threonine in nifH2 At position 102, arginine in the sequence Gly-Arg-Gly-Val-Ile-Thr might, as in Rhodospirillum rubrum, be the site of ‘switch-off’ inactivation. Compared with other nifH genes, the highest SAB was found with Bradyrhizobium japonicum and Rhizobium sp. (Parasponia). A sequence identical (nifH1) or very similar (nifH2) to the nif consensus sequence was found upstream from the initiation codon. Homology with sequences upstream from Klebsiella pneumoniae nif promoters, which affect nifA-mediated activation, was also found in both cases. From mutants carrying either a single nifH1 or nifH2 deletion, or a double nifH1-nifH2 deletion, it appeared that both genes were functional ex planta and in Planta and that no other nifH copy seemed to be present in ORS571. The optimal expression of nifH1 and of nifH2 might depend on different physiological conditions.