amoA Gene Abundances and Nitrification Potential Rates Suggest that Benthic Ammonia-Oxidizing Bacteria and Not Archaea Dominate N Cycling in the Colne Estuary,United Kingdom

Nitrification, mediated by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), is important in global nitrogen cycling. In estuaries where gradients of salinity and ammonia concentrations occur, there may be differential selections for ammonia-oxidizer populations. The aim of this study was to examine the activity, abundance, and diversity of AOA and AOB in surface oxic sediments of a highly nutrified estuary that exhibits gradients of salinity and ammonium. AOB and AOA communities were investigated by measuring ammonia monooxygenase (amoA) gene abundance and nitrification potentials both spatially and temporally. Nitrification potentials differed along the estuary and over time, with the greatest nitrification potentials occurring mid-estuary (8.2 μmol N grams dry weight [gdw]⁻¹ day⁻¹ in June, increasing to 37.4 μmol N gdw⁻¹ day⁻¹ in January). At the estuary head, the nitrification potential was 4.3 μmol N gdw⁻¹ day⁻¹ in June, increasing to 11.7 μmol N gdw⁻¹ day⁻¹ in January. At the estuary head and mouth, nitrification potentials fluctuated throughout the year. AOB amoA gene abundances were significantly greater (by 100-fold) than those of AOA both spatially and temporally. Nitrosomonas spp. were detected along the estuary by denaturing gradient gel electrophoresis (DGGE) band sequence analysis. In conclusion, AOB dominated over AOA in the estuarine sediments, with the ratio of AOB/AOA amoA gene abundance increasing from the upper (freshwater) to lower (marine) regions of the Colne estuary. These findings suggest that in this nutrified estuary, AOB (possibly Nitrosomonas spp.) were of major significance in nitrification.     

Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L⁻¹ for NO₃ ⁻ and NH₄ ⁺, 0.5–50 μmol N L⁻¹ for urea, 0.5–100 μmol N L⁻¹ for glu and cyanate, and 0.5–200 μmol N L⁻¹ for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μ_m, 1.51±0.06 d⁻¹ and 1.50±0.05 d⁻¹, respectively). However, cultures grown on cyanate, NO₃ ⁻, and NH₄ ⁺ had lower half saturation constants (K_μ, 0.28–0.51 μmol N L⁻¹). N uptake kinetics were measured in NO₃ ⁻-deplete and -replete batch cultures of P. donghaiense. In NO₃ ⁻-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO₃ ⁻, NH₄ ⁺, urea and algal amino acids; uptake was saturated at or below 50 μmol N L⁻¹. In NO₃ ⁻-replete batch cultures, NH₄ ⁺, urea, and algal amino acid uptake kinetics were similar to those measured in NO₃ ⁻-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.     

Low-Concentration Kinetics of Atmospheric CH4 Oxidation in Soil and Mechanism of NH4+ Inhibition

NH₄⁺ inhibition kinetics for CH₄ oxidation were examined at near-atmospheric CH₄ concentrations in three upland forest soils. Whether NH₄⁺-independent salt effects could be neutralized by adding nonammoniacal salts to control samples in lieu of deionized water was also investigated. Because the levels of exchangeable endogenous NH₄⁺ were very low in the three soils, desorption of endogenous NH₄⁺ was not a significant factor in this study. The K_m(app) values for water-treated controls were 9.8, 22, and 57 nM for temperate pine, temperate hardwood, and birch taiga soils, respectively. At CH₄ concentrations of ≤15 μl liter⁻¹, oxidation followed first-order kinetics in the fine-textured taiga soil, whereas the coarse-textured temperate soils exhibited Michaelis-Menten kinetics. Compared to water controls, the K_m(app) values in the temperate soils increased in the presence of NH₄⁺ salts, whereas the V_max(app) values decreased substantially, indicating that there was a mixture of competitive and noncompetitive inhibition mechanisms for whole NH₄⁺ salts. Compared to the corresponding K⁺ salt controls, the K_m(app) values for NH₄⁺ salts increased substantially, whereas the V_max(app) values remained virtually unchanged, indicating that NH₄⁺ acted by competitive inhibition. Nonammoniacal salts caused inhibition to increase with increasing CH₄ concentrations in all three soils. In the birch taiga soil, this trend occurred with both NH₄⁺ and K⁺ salts, and the slope of the increase was not affected by the addition of NH₄⁺. Hence, the increase in inhibition resulted from an NH₄⁺-independent mechanism. These results show that NH₄⁺ inhibition of atmospheric CH₄ oxidation resulted from enzymatic substrate competition and that additional inhibition that was not competitive resulted from a general salt effect that was independent of NH₄⁺.     

Source and Magnitude of Ammonium Generation in Maize Roots

Studies with ¹⁵N indicate that appreciable generation of NH₄⁺ from endogenous sources accompanies the uptake and assimilation of exogenous NH₄⁺ by roots. To identify the source of NH₄⁺ generation, maize (Zea mays L.) seedlings were grown on ¹⁴NH₄⁺ and then exposed for 3 d to highly labeled ¹⁵NH₄⁺. More of the entering ¹⁵NH₄⁺ was incorporated into the protein-N fraction of roots in darkness (approximately 25%) than in the light (approximately 14%). Although the ¹⁴NH₄⁺ content of roots declined rapidly to less than 1 μmol per plant, efflux of ¹⁴NH₄⁺ continued throughout the 3-d period at an average daily rate of 14 μmol per plant. As a consequence, cumulative ¹⁴NH₄⁺ efflux during the 3-d period accounted for 25% of the total ¹⁴N initially present in the root. Although soluble organic ¹⁴N in roots declined during the 3-d period, insoluble ¹⁴N remained relatively constant. In shoots both soluble organic ¹⁴N and ¹⁴NH₄⁺ declined, but a comparable increase in insoluble ¹⁴N was noted. Thus, total ¹⁴N in shoots remained constant, reflecting little or no net redistribution of ¹⁴N between shoots and roots. Collectively, these observations reveal that catabolism of soluble organic N, not protein N, is the primary source of endogenous NH₄⁺ generation in maize roots.     

Community Structure and Activity Dynamics of Nitrifying Bacteria in a Phosphate-Removing Biofilm

The microbial community structure and activity dynamics of a phosphate-removing biofilm from a sequencing batch biofilm reactor were investigated with special focus on the nitrifying community. O₂, NO₂⁻, and NO₃⁻ profiles in the biofilm were measured with microsensors at various times during the nonaerated-aerated reactor cycle. In the aeration period, nitrification was oxygen limited and restricted to the first 200 μm at the biofilm surface. Additionally, a delayed onset of nitrification after the start of the aeration was observed. Nitrate accumulating in the biofilm in this period was denitrified during the nonaeration period of the next reactor cycle. Fluorescence in situ hybridization (FISH) revealed three distinct ammonia-oxidizing populations, related to the Nitrosomonas europaea, Nitrosomonas oligotropha, and Nitrosomonas communis lineages. This was confirmed by analysis of the genes coding for 16S rRNA and for ammonia monooxygenase (amoA). Based upon these results, a new 16S rRNA-targeted oligonucleotide probe specific for the Nitrosomonas oligotropha lineage was designed. FISH analysis revealed that the first 100 μm at the biofilm surface was dominated by members of the N. europaea and the N. oligotropha lineages, with a minor fraction related to N. communis. In deeper biofilm layers, exclusively members of the N. oligotropha lineage were found. This separation in space and a potential separation of activities in time are suggested as mechanisms that allow coexistence of the different ammonia-oxidizing populations. Nitrite-oxidizing bacteria belonged exclusively to the genus Nitrospira and could be assigned to a 16S rRNA sequence cluster also found in other sequencing batch systems.     

Influence of starvation on potential ammonia-oxidizing activity and amoA mRNA levels of Nitrosospira briensis

The effect of short-term ammonia starvation on Nitrosospira briensis was investigated. The ammonia-oxidizing activity was determined in a concentrated cell suspension with a NOx biosensor. The apparent half-saturation constant [Km(app)] value of the NH3 oxidation of N. briensis was 3 microM NH3 for cultures grown both in continuous and batch cultures as determined by a NOx biosensor. Cells grown on the wall of the vessel had a lower Km(app) value of 1.8 microM NH3. Nonstarving cultures of N. briensis showed potential ammonia-oxidizing activities of between 200 to 250 microM N h(-1), and this activity decreased only slowly during starvation up to 10 days. Within 10 min after the addition of fresh NH4+, 100% activity was regained. Parallel with activity measurements, amoA mRNA and 16S rRNA were investigated. No changes were observed in the 16S rRNA, but a relative decrease of amoA mRNA was observed during the starvation period. During resuscitation, an increase in amoA mRNA expression was detected simultaneously. The patterns of the soluble protein fraction of a 2-week-starved culture of N. briensis showed only small differences in comparison to a nonstarved control. From these results we conclude that N. briensis cells remain in a state allowing fast recovery of ammonia-oxidizing activity after addition of NH4+ to a starved culture. Maintaining cells in this kind of active state could be the survival strategy of ammonia-oxidizing bacteria in nature under fluctuating NH4+ availability.     

In Situ Activity and Spatial Organization of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in Biofilms

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that “Brocadia”-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 μm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH₄⁺ and NO₂⁻ consumption rates decreased from 0.68 and 0.64 μmol cm⁻² h⁻¹ at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 μmol cm⁻² h⁻¹ at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH₄⁺ and NO₂⁻ and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O₂ or organic compounds, which consequently established suitable microenvironments for anammox bacteria.     

The Significance of Myriophyllum elatinoides for Swine Wastewater Treatment: Abundance and Community Structure of Ammonia-Oxidizing Microorganisms in Sediments

Myriophyllum elatinoides was reported to effectively treat wastewater by removing nitrogen (N) and phosphorus (P). However, little is known about the abundance and community structure of ammonia-oxidizing microorganisms associated with M. elatinoides purification systems. The objective of this research was to characterize the abundance and community structure of ammonia-oxidizing microorganisms in swine wastewater and determine the main nitrogen removal pathways. In this study, five different waters were treated by M. elatinoides in microcosms for one month. The five waters included tap water (Control), swine wastewater (SW), 50% diluted swine wastewater (50% SW), and two synthetic wastewaters: 200 mg NH₄ ⁺-N L⁻¹ (200 NH₄ ⁺-N) and 400 mg NH₄ ⁺-N L⁻¹ (400 NH₄ ⁺-N). The most dramatic changes were in NH₄ ⁺-N and total N (TN) concentrations, with average removal rates of 84% and 90%, respectively, in the treatments containing swine wastewater. On days 7, 14, and 28, the dissolved oxygen (DO) increased by 81.8%, 210.4% and 136.5%, respectively, compared with on day 0, in the swine wastewater. The results also showed that the bacterial amoA (AOB) copy numbers in the sediments of the treatments were significantly higher than those of archaeal amoA (AOA) copy numbers (p = 0.015). In addition, the high DO concentrations in swine wastewater responded well to the high abundance of AOB. The AOA and AOB community distributions were positively related with NO₃ ^-N and were negatively related with DO in swine wastewater treatments. In summary, our experimental results suggested that the M. elatinoides purification system could improve the activity of ammonia-oxidizing microorganisms and consequently might contribute to the significant N removal from the swine wastewater.     

Steady-State Growth and Chemical Composition of the Marine Chlorophyte Dunaliella tertiolecta in Nitrogen-Limited Continuous Cultures

The marine chlorophyte Dunaliella tertiolecta was grown in continuous cultures under NH₄⁺-N, NO₂⁻-N, NO₃⁻-N, and urea-N limitations. The effect of the nitrogen cell quota (Q_n) on the steady-state growth rate (μ) was the same regardless of the N source. The relationship between μ and Q_n was well described by the Droop equation, but only up to the true maximum growth rate ^μ (= cell washout rate). The ratio between the minimum cell quota (k_Q) and the maximum cell quota (Q_m) was 0.19. Hence, there is no substitute for determining ^μ experimentally. That there was no difference in growth response to different N sources suggests that no internal pooling of inorganic nitrogen occurred. Both the carbon (Q_c) and phosphorus (Q_p) cell quotas under N limitation increased with increasing μ in a threshold fashion: virtually no change in either cell quota up to ~0.8 ^μ, followed by a rapid and large increase up to ^μ. In addition, in the region of low μ, there was an increase in Q_p with a decreasing medium N/P ratio of between 15 and 5 (by atoms). The results generally indicate the physiological limits in cellular constituency under N limitation. The usefulness of this information, however, in describing the response of natural populations of marine phytoplankton to transient nutrient exposures on the temporal and spatial microscales that most likely exist is of limited value.     

New Insights into Methyl Bromide Cooxidation by Nitrosomonas europaea Obtained by Experimenting with Moderately Low Density Cell Suspensions

We examined the rates and sustainability of methyl bromide (MeBr) oxidation in moderately low density cell suspensions (~6 × 10⁷ cells ml⁻¹) of the NH₃-oxidizing bacterium Nitrosomonas europaea. In the presence of 10 mM NH₄⁺ and 0.44, 0.22, and 0.11 mM MeBr, the initial rates of MeBr oxidation were sustained for 12, 12, and 24 h, respectively, despite the fact that only 10% of the NH₄⁺, 18% of the NH₄⁺, and 35% of the NH₄⁺, respectively, were consumed. Although the duration of active MeBr oxidation generally decreased as the MeBr concentration increased, similar amounts of MeBr were oxidized with a large number of the NH₄⁺-MeBr combinations examined (10 to 20 μmol mg [dry weight] of cells⁻¹). Approximately 90% of the NH₃-dependent O₂ uptake activity and the NO₂⁻-producing activity were lost after N. europaea was exposed to 0.44 mM MeBr for 24 h. After MeBr was removed and the cells were resuspended in fresh growth medium, NO₂⁻ production increased exponentially, and 48 to 60 h was required to reach the level of activity observed initially in control cells that were not exposed to MeBr. It is not clear what percentage of the cells were capable of cell division after MeBr oxidation because NO₂⁻ accumulated more slowly in the exposed cells than in the unexposed cells despite the fact that the latter were diluted 10-fold to create inocula which exhibited equal initial activities. The decreases in NO₂⁻-producing and MeBr-oxidizing activities could not be attributed directly to NH₄⁺ or NH₃ limitation, to a decrease in the pH, to the composition of the incubation medium, or to toxic effects caused by accumulation of the end products of oxidation (NO₂⁻ and formaldehyde) in the medium. Additional cooxidation-related studies of N. europaea are needed to identify the mechanism(s) responsible for the MeBr-induced loss of cell activity and/or viability, to determine what percentages of cells damaged by cooxidative activities are culturable, and to determine if cooxidative activity interferes with the regulation of NH₃-oxidizing activity.     

Bioinformatic and Expression Analyses of Genes Mediating Zinc Homeostasis in Nostoc punctiforme

Zinc homeostasis was investigated in Nostoc punctiforme. Cell tolerance to Zn²⁺ over 14 days showed that ZnCl₂ levels above 22 μM significantly reduced cell viability. After 3 days in 22 μM ZnCl₂, ca. 12% of the Zn²⁺ was in an EDTA-resistant component, suggesting an intracellular localization. Zinquin fluorescence was detected within cells exposed to concentrations up to 37 μM relative to 0 μM treatment. Radiolabeled ⁶⁵Zn showed Zn²⁺ uptake increased over a 3-day period, while efflux occurred more rapidly within a 3-h time period. Four putative genes involved in Zn²⁺ uptake and efflux in N. punctiforme were identified: (i) the predicted Co/Zn/Cd cation transporter, putative CDF; (ii) the predicted divalent heavy-metal cation transporter, putative Zip; (iii) the ATPase component and Fe/Zn uptake regulation protein, putative Fur; and (iv) an ABC-type Mn/Zn transport system, putative zinc ZnuC, ZnuABC system component. Quantitative real-time PCR indicated the responsiveness of all four genes to 22 μM ZnCl₂ within 3 h, followed by a reduction to below basal levels after 24 h by putative ZIP, ZnuC, and Fur and a reduction to below basal level after 72 h by putative CDF efflux gene. These results demonstrate differential regulation of zinc transporters over time, indicating a role for them in zinc homeostasis in N. punctiforme.     

Inhibitory Effects of C2 to C10 1-Alkynes on Ammonia Oxidation in Two Nitrososphaera Species

A previous study showed that ammonia oxidation by the Thaumarchaeota Nitrosopumilus maritimus (group 1.1a) was resistant to concentrations of the C₈ 1-alkyne, octyne, which completely inhibits activity by ammonia-oxidizing bacteria. In this study, the inhibitory effects of octyne and other C₂ to C₁₀ 1-alkynes were evaluated on the nitrite production activity of two pure culture isolates from Thaumarchaeota group 1.1b, Nitrososphaera viennensis strain EN76 and Nitrososphaera gargensis. Both N. viennensis and N. gargensis were insensitive to concentrations of octyne that cause complete and irreversible inactivation of nitrite production by ammonia-oxidizing bacteria. However, octyne concentrations (≥20 μM) that did not inhibit N. maritimus partially inhibited nitrite production in N. viennensis and N. gargensis in a manner that did not show the characteristics of irreversible inactivation. In contrast to previous studies with an ammonia-oxidizing bacterium, Nitrosomonas europaea, octyne inhibition of N. viennensis was: (i) fully and immediately reversible, (ii) not competitive with NH₄⁺, and (iii) without effect on the competitive interaction between NH₄⁺ and acetylene. Both N. viennensis and N. gargensis demonstrated the same overall trend in regard to 1-alkyne inhibition as previously observed for N. maritimus, being highly sensitive to ≤C₅ alkynes and more resistant to longer-chain length alkynes. Reproducible differences were observed among N. maritimus, N. viennensis, and N. gargensis in regard to the extent of their resistance/sensitivity to C₆ and C₇ 1-alkynes, which may indicate differences in the ammonia monooxygenase binding and catalytic site(s) among the Thaumarchaeota.     

Effect of Soil Ammonium Concentration on N2O Release and on the Community Structure of Ammonia Oxidizers and Denitrifiers

The effect of ammonium addition (6.5, 58, and 395 μg of NH₄⁺-N g [dry weight] of soil⁻¹) on soil microbial communities was explored. For medium and high ammonium concentrations, increased N₂O release rates and a shift toward a higher contribution of nitrification to N₂O release occurred after incubation for 5 days at 4°C. Communities of ammonia oxidizers were assayed after 4 weeks of incubation by denaturant gradient gel electrophoresis (DGGE) of the amoA gene coding for the small subunit of ammonia monooxygenase. The DGGE fingerprints were invariably the same whether the soil was untreated or incubated with low, medium, or high ammonium concentrations. Phylogenetic analysis of cloned PCR products from excised DGGE bands detected amoA sequences which probably belonged to Nitrosospira 16S rRNA clusters 3 and 4. Additional clones clustered with Nitrosospira sp. strains Ka3 and Ka4 and within an amoA cluster from unknown species. A Nitrosomonas-like amoA gene was detected in only one clone. In agreement with the amoA results, community profiles of total bacteria analyzed by terminal restriction fragment length polymorphism (T-RFLP) showed only minor differences. However, a community shift occurred for denitrifier populations based on T-RFLP analysis of nirK genes encoding copper-containing nitrite reductase with incubation at medium and high ammonia concentrations. Major terminal restriction fragments observed in environmental samples were further described by correspondence to cloned nirK genes from the same soil. Phylogenetic analysis grouped these clones into clusters of soil nirK genes. However, some clones were also closely related to genes from known denitrifiers. The shift in the denitrifier community was probably the consequence of the increased supply of oxidized nitrogen through nitrification. Nitrification activity increased upon addition of ammonium, but the community structure of ammonium oxidizers did not change.     

Anaerobic Ammonia Oxidation in the Presence of Nitrogen Oxides (NOx) by Two Different Lithotrophs

The anaerobic ammonia-oxidizing activity of the planctomycete Candidatus “Brocadia anammoxidans” was not inhibited by NO concentrations up to 600 ppm and NO₂ concentrations up to 100 ppm. B. anammoxidans was able to convert (detoxify) NO, which might explain the high NO tolerance of this organism. In the presence of NO₂, the specific ammonia oxidation activity of B. anammoxidans increased, and Nitrosomonas-like microorganisms recovered an NO₂-dependent anaerobic ammonia oxidation activity. Addition of NO₂ to a mixed population of B. anammoxidans and Nitrosomonas induced simultaneous specific anaerobic ammonia oxidation activities of up to 5.5 mmol of NH₄⁺ g of protein⁻¹ h⁻¹ by B. anammoxidans and up to 1.5 mmol of NH₄⁺ g of protein⁻¹ h⁻¹ by Nitrosomonas. The stoichiometry of the converted N compounds (NO₂⁻/NH₃ ratio) and the microbial community structure were strongly influenced by NO₂. The combined activity of B. anammoxidans and Nitrosomonas-like ammonia oxidizers might be of relevance in natural environments and for technical applications.     

A Cytochrome cbb3 (Cytochrome c) Terminal Oxidase in Azospirillum brasilense Sp7 Supports Microaerobic Growth

Spectral analysis indicated the presence of a cytochrome cbb₃ oxidase under microaerobic conditions in Azospirillum brasilense Sp7 cells. The corresponding genes (cytNOQP) were isolated by using PCR. These genes are organized in an operon, preceded by a putative anaerobox. The phenotype of an A. brasilense cytN mutant was analyzed. Under aerobic conditions, the specific growth rate during exponential phase (μ_e) of the A. brasilense cytN mutant was comparable to the wild-type specific growth rate (μ_e of approximately 0.2 h⁻¹). In microaerobic NH₄⁺-supplemented conditions, the low respiration of the A. brasilense cytN mutant affected its specific growth rate (μ_e of approximately 0.02 h⁻¹) compared to the wild-type specific growth rate (μ_e of approximately 0.2 h⁻¹). Under nitrogen-fixing conditions, both the growth rates and respiration of the wild type were significantly diminished in comparison to those under NH₄⁺-supplemented conditions. Differences in growth rates and respiration between the wild type and the A. brasilense cytN mutant were less pronounced under these nitrogen-fixing conditions (μ_e of approximately 0.03 h⁻¹ for the wild type and 0.02 h⁻¹ for the A. brasilense cytN mutant). The nitrogen-fixing capacity of the A. brasilense cytN mutant was still approximately 80% of that determined for the wild-type strain. This leads to the conclusion that the A. brasilense cytochrome cbb₃ oxidase is required under microaerobic conditions, when a high respiration rate is needed, but that under nitrogen-fixing conditions the respiration rate does not seem to be a growth-limiting factor.     

Use of Aliphatic n-Alkynes To Discriminate Soil Nitrification Activities of Ammonia-Oxidizing Thaumarchaea and Bacteria

Ammonia (NH₃)-oxidizing bacteria (AOB) and thaumarchaea (AOA) co-occupy most soils, yet no short-term growth-independent method exists to determine their relative contributions to nitrification in situ. Microbial monooxygenases differ in their vulnerability to inactivation by aliphatic n-alkynes, and we found that NH₃ oxidation by the marine thaumarchaeon Nitrosopumilus maritimus was unaffected during a 24-h exposure to ≤20 μM concentrations of 1-alkynes C₈ and C₉. In contrast, NH₃ oxidation by two AOB (Nitrosomonas europaea and Nitrosospira multiformis) was quickly and irreversibly inactivated by 1 μM C₈ (octyne). Evidence that nitrification carried out by soilborne AOA was also insensitive to octyne was obtained. In incubations (21 or 28 days) of two different whole soils, both acetylene and octyne effectively prevented NH₄⁺-stimulated increases in AOB population densities, but octyne did not prevent increases in AOA population densities that were prevented by acetylene. Furthermore, octyne-resistant, NH₄⁺-stimulated net nitrification rates of 2 and 7 μg N/g soil/day persisted throughout the incubation of the two soils. Other evidence that octyne-resistant nitrification was due to AOA included (i) a positive correlation of octyne-resistant nitrification in soil slurries of cropped and noncropped soils with allylthiourea-resistant activity (100 μM) and (ii) the finding that the fraction of octyne-resistant nitrification in soil slurries correlated with the fraction of nitrification that recovered from irreversible acetylene inactivation in the presence of bacterial protein synthesis inhibitors and with the octyne-resistant fraction of NH₄⁺-saturated net nitrification measured in whole soils. Octyne can be useful in short-term assays to discriminate AOA and AOB contributions to soil nitrification.     

Genetic disruption of Abl nuclear import reduces renal apoptosis in a mouse model of cisplatin-induced nephrotoxicity

DNA damage activates nuclear Abl tyrosine kinase to stimulate intrinsic apoptosis in cancer cell lines and mouse embryonic stem cells. To examine the in vivo function of nuclear Abl in apoptosis, we generated Abl-μNLS (μ, mutated in nuclear localization signals) mice. We show here that cisplatin-induced apoptosis is defective in the renal proximal tubule cells (RPTC) from the Abl^μ/μ mice. When injected with cisplatin, we found similar levels of platinum in the Abl^+/+ and the Abl^μ/μ kidneys, as well as similar initial inductions of p53 and PUMAα expression. However, the accumulation of p53 and PUMAα could not be sustained in the Abl^μ/μ kidneys, leading to reductions in renal apoptosis and tubule damage. Co-treatment of cisplatin with the Abl kinase inhibitor, imatinib, reduced the accumulation of p53 and PUMAα in the Abl^+/+ but not in the Abl^μ/μ kidneys. The residual apoptosis in the Abl^μ/μ mice was not further reduced in the Abl^μ/μ; p53^−/− double-mutant mice, suggesting that nuclear Abl and p53 are epistatic to each other in this apoptosis response. Although apoptosis and tubule damage were reduced, cisplatin-induced increases in phospho-Stat-1 and blood urea nitrogen were similar between the Abl^+/+ and the Abl^μ/μ kidneys, indicating that RPTC apoptosis is not the only factor in cisplatin-induced nephrotoxicity. These results provide in vivo evidence for the pro-apoptotic function of Abl, and show that its nuclear localization and tyrosine kinase activity are both required for the sustained expression of p53 and PUMAα in cisplatin-induced renal apoptosis.     

Loss of Ammonia Monooxygenase Activity in Nitrosomonas europaea upon Exposure to Nitrite

Nitrosomonas europaea, an obligate ammonia-oxidizing bacterium, lost an increasing amount of ammonia oxidation activity upon exposure to increasing concentrations of nitrite, the primary product of ammonia-oxidizing metabolism. The loss of activity was specific to the ammonia monooxygenase (AMO) enzyme, as confirmed by a decreased rate of NH₄⁺-dependent O₂ consumption, some loss of active AMO molecules observed by polypeptide labeling with ¹⁴C₂H₂, the protection of activity by substrates of AMO, and the requirement for copper. The loss of AMO activity via nitrite occurred under both aerobic and anaerobic conditions, and more activity was lost under alkaline than under acidic conditions except in the presence of large concentrations (20 mM) of nitrite. These results indicate that nitrite toxicity in N. europaea is mediated by a unique mechanism that is specific for AMO.     

Purification, Characterization, and Gene Cloning of Purine Nucleosidase from Ochrobactrum anthropi

A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible N-riboside hydrolysis of purine nucleosides, the K_m values being 11.8 to 56.3 μM. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with K_i and K_i′ values of 0.455 to 11.2 μM. Metal ion chelators inhibited activity, and the addition of Zn²⁺ or Co²⁺ restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in Escherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH₂-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.