48 research outputs found
Role of a Fur homolog in iron metabolism in Nitrosomonas europaea
<p>Abstract</p> <p>Background</p> <p>In response to environmental iron concentrations, many bacteria coordinately regulate transcription of genes involved in iron acquisition via the ferric uptake regulation (Fur) system. The genome of <it>Nitrosomonas europaea</it>, an ammonia-oxidizing bacterium, carries three genes (NE0616, NE0730 and NE1722) encoding proteins belonging to Fur family.</p> <p>Results</p> <p>Of the three <it>N. europaea fur </it>homologs, only the Fur homolog encoded by gene NE0616 complemented the <it>Escherichia coli </it>H1780 <it>fur </it>mutant. A <it>N. europaea fur:kanP </it>mutant strain was created by insertion of kanamycin-resistance cassette in the promoter region of NE0616 <it>fur </it>homolog. The total cellular iron contents of the <it>fur:kanP </it>mutant strain increased by 1.5-fold compared to wild type when grown in Fe-replete media. Relative to the wild type, the <it>fur:kanP </it>mutant exhibited increased sensitivity to iron at or above 500 μM concentrations. Unlike the wild type, the <it>fur:kanP </it>mutant was capable of utilizing iron-bound ferrioxamine without any lag phase and showed over expression of several outer membrane TonB-dependent receptor proteins irrespective of Fe availability.</p> <p>Conclusions</p> <p>Our studies have clearly indicated a role in Fe regulation by the Fur protein encoded by <it>N. europaea </it>NE0616 gene. Additional studies are required to fully delineate role of this <it>fur </it>homolog.</p
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Steady-State Growth under Inorganic Carbon Limitation Conditions Increases Energy Consumption for Maintenance and Enhances Nitrous Oxide Production in Nitrosomonas europaea
Nitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH₃) to obtain energy for growth on carbon dioxide (CO₂) and can also produce nitrous oxide (N₂O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses of N. europaea to conditions of replete (>5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na₂CO₃) supplemented with atmospheric CO₂. IC-limited cultures oxidized 25 to 58% of available NH₃ to nitrite, depending on the dilution rate and Na₂CO₃ concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to those for NH₃-limited cultures. Rates of N₂O production increased 2.5- and 6.3-fold under the two IC-limited conditions, increasing the percentage of oxidized NH₃-N that was transformed to N₂O-N from 0.5% (replete) up to 4.4% (0.2 mM Na₂CO₃). Transcriptome analysis showed differential expression (P ≤ 0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in a decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits and increased the expression of genes involved in C₁ metabolism, including the genes for RuBisCO (cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C₁ moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924 to NE0927) correlated with increased production of N₂O. Together, these data suggest that N. europaea adapts physiologically during IC-limited steady-state growth, which leads to the uncoupling of NH₃ oxidation from growth and increased N₂O production.
IMPORTANCE: Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, is an important process in the global nitrogen cycle. This process is generally dependent on ammonia-oxidizing microorganisms and nitrite-oxidizing bacteria. Most nitrifiers are chemolithoautotrophs that fix inorganic carbon (CO₂) for growth. Here, we investigate how inorganic carbon limitation modifies the physiology and transcriptome of Nitrosomonas europaea, a model ammonia-oxidizing bacterium, and report on increased production of N₂O, a potent greenhouse gas. This study, along with previous work, suggests that inorganic carbon limitation may be an important factor in controlling N₂O emissions from nitrification in soils and wastewater treatment
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Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly ‘Pseudomonas butanovora’
Soluble butane monooxygenase (sBMO), a three-component di-iron monooxygenase complex
expressed by the C2–C9 alkane-utilizing bacterium Thauera butanivorans, was kinetically
characterized by measuring substrate specificities for C1–C5 alkanes and product inhibition
profiles. sBMO has high sequence homology with soluble methane monooxygenase (sMMO) and
shares a similar substrate range, including gaseous and liquid alkanes, aromatics, alkenes and
halogenated xenobiotics. Results indicated that butane was the preferred substrate (defined by
kcat : Km ratios). Relative rates of oxidation for C1–C5 alkanes differed minimally, implying that
substrate specificity is heavily influenced by differences in substrate Km values. The low
micromolar Km for linear C2–C5 alkanes and the millimolar Km for methane demonstrate that
sBMO is two to three orders of magnitude more specific for physiologically relevant substrates of
T. butanivorans. Methanol, the product of methane oxidation and also a substrate itself, was found
to have similar Km and kcat values to those of methane. This inability to kinetically discriminate
between the C1 alkane and C1 alcohol is observed as a steady-state concentration of methanol
during the two-step oxidation of methane to formaldehyde by sBMO. Unlike methanol, alcohols
with chain length C2–C5 do not compete effectively with their respective alkane substrates.
Results from product inhibition experiments suggest that the geometry of the active site is
optimized for linear molecules four to five carbons in length and is influenced by the regulatory
protein component B (butane monooxygenase regulatory component; BMOB). The data suggest
that alkane oxidation by sBMO is highly specialized for the turnover of C3–C5 alkanes and the
release of their respective alcohol products. Additionally, sBMO is particularly efficient at
preventing methane oxidation during growth on linear alkanes ≥C2, despite its high sequence
homology with sMMO. These results represent, to the best of our knowledge, the first kinetic in
vitro characterization of the closest known homologue of sMM
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Identifying Potential Mechanisms Enabling Acidophily in the Ammonia-Oxidizing Archaeon "Candidatus Nitrosotalea devanaterra"
Ammonia oxidation is the first and rate-limiting step in nitrification and is dominated by two distinct groups of microorganisms in soil: ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). AOA are often more abundant than AOB and dominate activity in acid soils. The mechanism of ammonia oxidation under acidic conditions has been a long-standing paradox. While high rates of ammonia oxidation are frequently measured in acid soils, cultivated ammonia oxidizers grew only at near-neutral pH when grown in standard laboratory culture. Although a number of mechanisms have been demonstrated to enable neutrophilic AOB growth at low pH in the laboratory, these have not been demonstrated in soil, and the recent cultivation of the obligately acidophilic ammonia oxidizer “Candidatus Nitrosotalea devanaterra” provides a more parsimonious explanation for the observed high rates of activity. Analysis of the sequenced genome, transcriptional activity, and lipid content of “Ca. Nitrosotalea devanaterra” reveals that previously proposed mechanisms used by AOB for growth at low pH are not essential for archaeal ammonia oxidation in acidic environments. Instead, the genome indicates that “Ca. Nitrosotalea devanaterra” contains genes encoding both a predicted high-affinity substrate acquisition system and potential pH homeostasis mechanisms absent in neutrophilic AOA. Analysis of mRNA revealed that candidate genes encoding the proposed homeostasis mechanisms were all expressed during acidophilic growth, and lipid profiling by high-performance liquid chromatography–mass spectrometry (HPLC-MS) demonstrated that the membrane lipids of “Ca. Nitrosotalea devanaterra” were not dominated by crenarchaeol, as found in neutrophilic AOA. This study for the first time describes a genome of an obligately acidophilic ammonia oxidizer and identifies potential mechanisms enabling this unique phenotype for future biochemical characterization
Ca. Nitrososphaera and Bradyrhizobium are inversely correlated and related to agricultural practices in long-term field experiments
Agricultural land management, such as fertilization, liming, and tillage affects soil properties, including pH, organic matter content, nitrification rates, and the microbial community. Three different study sites were used to identify microorganisms that correlate with agricultural land use and to determine which factors regulate the relative abundance of the microbial signatures of the agricultural land-use. The three sites included in this study are the Broadbalk Experiment at Rothamsted Research, UK, the Everglades Agricultural Area, Florida, USA, and the Kellogg Biological Station, Michigan, USA. The effects of agricultural management on the abundance and diversity of bacteria and archaea were determined using high throughput, barcoded 16S rRNA sequencing. In addition, the relative abundance of these organisms was correlated with soil features. Two groups of microorganisms involved in nitrogen cycle were highly correlated with land use at all three sites. The ammonia oxidizing-archaea, dominated by Ca. Nitrososphaera, were positively correlated with agriculture while a ubiquitous group of soil bacteria closely related to the diazotrophic symbiont, Bradyrhizobium, was negatively correlated with agricultural management. Analysis of successional plots showed that the abundance of ammonia oxidizing-archaea declined and the abundance of bradyrhizobia increased with time away from agriculture. This observation suggests that the effect of agriculture on the relative abundance of these genera is reversible. Soil pH and NH(3) concentrations were positively correlated with archaeal abundance but negatively correlated with the abundance of Bradyrhizobium. The high correlations of Ca. Nitrososphaera and Bradyrhizobium abundances with agricultural management at three long-term experiments with different edaphoclimatic conditions allowed us to suggest these two genera as signature microorganisms for agricultural land use
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Complete genome sequence of Nitrosomonas sp. Is79, an ammonia oxidizing bacterium adapted to low ammonium concentrations
Nitrosomonas sp. Is79 is a chemolithoautotrophic ammonia-oxidizing bacterium that belongs to the family Nitrosomonadaceae within the phylum Proteobacteria. Ammonia oxidation is the first step of nitrification, an important process in the global nitrogen cycle ultimately resulting in the production of nitrate. Nitrosomonas sp. Is79 is an ammonia oxidizer of high interest because it is adapted to low ammonium and can be found in freshwater environments around the world. The 3,783,444-bp chromosome with a total of 3,553 protein coding genes and 44 RNA genes was sequenced by the DOE-Joint Genome Institute Program CSP 2006.Keywords: nitrification,
Ammonia-oxidizing bacteria,
Ammonia oxidation,
Nitrosomonas,
nitrogen cycle,
oligotrophic,
freshwate
Alkane Utilization by Rhodococcus Strain NTU-1 Alone and in Its Natural Association with Bacillus fusiformis L-l and Ochrobactrum sp.
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Metabolomic Functional Analysis of Bacterial Genomes: Final Report
The availability of the complete DNA sequence of the bacterial genome of Nitrosomonas europaea offered the opportunity for unprecedented and detailed investigations of function. We studied the function of genes involved in carbohydrate and Fe metabolism. N. europaea has genes for the synthesis and degradation of glycogen and sucrose but cannot grow on substrates other than ammonia and CO2. Granules of glycogen were detected in whole cells by electron microscopy and quantified in cell-free extracts by enzymatic methods. The cellular glycogen and sucrose content varied depending on the composition of the growth medium and cellular growth stage. N. europaea also depends heavily on iron for metabolism of ammonia, is particularly interesting since it lacks genes for siderophore production, and has genes with only low similarity to known iron reductases, yet grows relatively well in medium containing low Fe. By comparing the transcriptomes of cells grown in iron-replete medium versus iron-limited medium, 247 genes were identified as differentially expressed. Mutant strains deficient in genes for sucrose, glycogen and iron metabolism were created and are being used to further our understanding of ammonia oxidizing bacteria