Jeanette M. Norton

Complete Genome Sequence of the Ammonia-Oxidizing Bacterium and Obligate Chemolithoautotroph Nitrosomonas europaea

Nitrosomonas europaea (ATCC 19718) is a gram-negative obligate chemolithoautotroph that can derive all its energy and reductant for growth from the oxidation of ammonia to nitrite. Nitrosomonas europaea participates in the biogeochemical N cycle in the process of nitrification. Its genome consists of a single circular chromosome of 2,812,094 bp. The GC skew analysis indicates that the genome is divided into two unequal replichores. Genes are distributed evenly around the genome, with approximately 47% transcribed from one strand and approximately 53% transcribed from the complementary strand. A total of 2,460 protein-encoding genes emerged from the modeling effort, averaging 1,011 bp in length, with intergenic regions averaging 117 bp. Genes necessary for the catabolism of ammonia, energy and reductant generation, biosynthesis, and CO(2) and NH(3) assimilation were identified. In contrast, genes for catabolism of organic compounds are limited. Genes encoding transporters for inorganic ions were plentiful, whereas genes encoding transporters for organic molecules were scant. Complex repetitive elements constitute ca. 5% of the genome. Among these are 85 predicted insertion sequence elements in eight different families. The strategy of N. europaea to accumulate Fe from the environment involves several classes of Fe receptors with more than 20 genes devoted to these receptors. However, genes for the synthesis of only one siderophore, citrate, were identified in the genome. This genome has provided new insights into the growth and metabolism of ammonia-oxidizing bacteria.

Diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria

Abstract.Autotrophic ammonia-oxidizing bacteria use the essential enzyme ammonia monooxygenase (AMO) to transform ammonia to hydroxylamine. The amo operon consists of at least three genes, amoC, amoA, and amoB; amoA encodes the subunit containing the putative enzyme active site. The use of the amo genes as functional markers for ammonia-oxidizing bacteria in environmental applications requires knowledge of the diversity of the amo operon on several levels: (1) the copy number of the operon in the genome, (2) the arrangement of the three genes in an individual operon, and (3) the primary sequence of the individual genes. We present a database of amo gene sequences for pure cultures of ammonia-oxidizing bacteria representing both the β- and the γ-subdivision of Proteobacteria in the following genera: Nitrosospira (6 strains), Nitrosomonas (5 strains) and Nitrosococcus (2 strains). The amo operon was found in multiple (2–3) nearly identical copies in the β-subdivision representatives but in single copies in the γ-subdivision ammonia oxidizers. The analysis of the deduced amino acid sequence revealed strong conservation for all three Amo peptides in both primary and secondary structures. For the amoA gene within the β-subdivision, nucleotide identity values are approximately 85% within the Nitrosomonas or the Nitrosospira groups, but approximately 75% when comparing between these groups. Conserved regions in amoA and amoC were identified and used as primer sites for PCR amplification of amo genes from pure cultures, enrichments and the soil environment. The intergenic region between amoC and amoA is variable in length and may be used to profile the community of ammonia-oxidizing bacteria in environmental samples. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-001-0369-z.

N dynamics in the rhizosphere of Pinus ponderosa seedlings

Transformations of inorganic N were studied in microcosms containing Pinus ponderosa seedlings. We assessed the potential for immobilization and mineralization of NH4+ in soils collected from adjacent to coarse, fine and young roots and in soil more than 5 mm from any root. Rates of mineralization and immobilization of NH4+ in soils collected from adjacent to roots were >50% higher than those in soils more than 5 mm from any root. C input estimates suggest that soils adjacent to fine or young roots could have supported immobilization rates higher than those observed. We determined the response of mineralization and immobilization rates to increased NH4+ in rhizosphere and bulk soil. Increasing NH4+ addition resulted in proportional increases in immobilization rates in both rhizosphere and bulk soils but did not affect mineralization rates. The increased immobilization rates with increasing NH4+ addition and the failure to predict immobilization rates based on C supply suggested that in the short-term, immobilization rates were limited by NH4+ supply rather than C availability. In intact microcosms, we determined the transformation rates of inorganic N using a combined 15N pool dilution and tracer approach. The rates of mineralization, immobilization of NH4+ and NO3−, nitrification and plant uptake were determined over 48 h. Rates in the root-zone were compared to rates in soils from which roots had been excluded for 2 weeks before labeling. The elimination of active roots from soil regions did not significantly change the production or the total consumption of NH4+ in those regions. The presence of roots reduced microbial consumption of NH4+ by nitrifiers and heterotrophs. Pine roots were successful competitors with microorganisms for limited inorganic N, but were more successful when the N source was NO3− vs NH4+. Plants accounted for 30% of the total NH4+ consumption, but 70% of total NO3− consumption.

Complete Genome Sequence of Nitrosospira multiformis, an Ammonia-Oxidizing Bacterium from the Soil Environment

ABSTRACT The complete genome of the ammonia-oxidizing bacterium Nitrosospira multiformis (ATCC 25196T) consists of a circular chromosome and three small plasmids totaling 3,234,309 bp and encoding 2,827 putative proteins. Of the 2,827 putative proteins, 2,026 proteins have predicted functions and 801 are without conserved functional domains, yet 747 of these have similarity to other predicted proteins in databases. Gene homologs from Nitrosomonas europaea and Nitrosomonas eutropha were the best match for 42% of the predicted genes in N. multiformis. The N. multiformis genome contains three nearly identical copies of amo and hao gene clusters as large repeats. The features of N. multiformis that distinguish it from N. europaea include the presence of gene clusters encoding urease and hydrogenase, a ribulose-bisphosphate carboxylase/oxygenase-encoding operon of distinctive structure and phylogeny, and a relatively small complement of genes related to Fe acquisition. Systems for synthesis of a pyoverdine-like siderophore and for acyl-homoserine lactone were unique to N. multiformis among the sequenced genomes of ammonia-oxidizing bacteria. Gene clusters encoding proteins associated with outer membrane and cell envelope functions, including transporters, porins, exopolysaccharide synthesis, capsule formation, and protein sorting/export, were abundant. Numerous sensory transduction and response regulator gene systems directed toward sensing of the extracellular environment are described. Gene clusters for glycogen, polyphosphate, and cyanophycin storage and utilization were identified, providing mechanisms for meeting energy requirements under substrate-limited conditions. The genome of N. multiformis encodes the core pathways for chemolithoautotrophy along with adaptations for surface growth and survival in soil environments.

Complete Genome Sequence of the Marine, Chemolithoautotrophic, Ammonia-Oxidizing Bacterium Nitrosococcus oceani ATCC 19707

ABSTRACT The gammaproteobacterium Nitrosococcus oceani (ATCC 19707) is a gram-negative obligate chemolithoautotroph capable of extracting energy and reducing power from the oxidation of ammonia to nitrite. Sequencing and annotation of the genome revealed a single circular chromosome (3,481,691 bp; G+C content of 50.4%) and a plasmid (40,420 bp) that contain 3,052 and 41 candidate protein-encoding genes, respectively. The genes encoding proteins necessary for the function of known modes of lithotrophy and autotrophy were identified. Contrary to betaproteobacterial nitrifier genomes, the N. oceani genome contained two complete rrn operons. In contrast, only one copy of the genes needed to synthesize functional ammonia monooxygenase and hydroxylamine oxidoreductase, as well as the proteins that relay the extracted electrons to a terminal electron acceptor, were identified. The N. oceani genome contained genes for 13 complete two-component systems. The genome also contained all the genes needed to reconstruct complete central pathways, the tricarboxylic acid cycle, and the Embden-Meyerhof-Parnass and pentose phosphate pathways. The N. oceani genome contains the genes required to store and utilize energy from glycogen inclusion bodies and sucrose. Polyphosphate and pyrophosphate appear to be integrated in this bacteriums energy metabolism, stress tolerance, and ability to assimilate carbon via gluconeogenesis. One set of genes for type I ribulose-1,5-bisphosphate carboxylase/oxygenase was identified, while genes necessary for methanotrophy and for carboxysome formation were not identified. The N. oceani genome contains two copies each of the genes or operons necessary to assemble functional complexes I and IV as well as ATP synthase (one H+-dependent F0F1 type, one Na+-dependent V type).

Contrasting responses of Intermountain West grasses to soil nitrogen

The mechanisms responsible for soil-N-mediated species replacement of native perennial grasses by the invasive annual grasses cheatgrass (Bromus tectorum L.) and medusahead (Taeniatherum caput-medusae [L.] Nevski) on rangelands are not completely understood. In addition, the contributions of distinct forms of inorganic N (i.e., NH 4 + and NO 3 -) to these shifts in species composition are currently unclear. Consequently, we conducted a greenhouse experiment to test 2 hypotheses: 1) that low N availability reduces growth (root and shoot) and N allocation of invasive annual seedlings more than native perennial species, and 2) that seedling growth and N allocation of invasive annual grasses is more responsive than native perennial grasses when supplied with NO 3 - relative to NH 4 +. We grew seedlings of 2 annual grasses and the native perennial grasses bluebunch wheatgrass (Pseudoroegneria spicata [Pursh] A. Love), and 4 populations of squirreltail (Elymus elymoides [Raf.] Swezey; E. multisetus [J.G. Smith] M.E. Jones) in separate pots and exposed them to treatments differing in N form and availability for 17 weeks. Unexpectedly, root and shoot growth of annual grasses were equal or greater than native perennial grasses under low N availability. Annual grasses took up more NO 3 - and allocated more growth and N to shoots than the perennial grasses (P < 0.05). Perennial grasses had significantly greater root:shoot dry mass ratios than the invasive annual grasses across treatments (P < 0.05). Invasive annual and native perennial grasses both had greater (P < 0.05) shoot and root mass and allocated more N to these structures when supplied with NO 3 - relative to NH 4 +. The ecological implications of these growth and N allocation patterns in response to N availability and form provide important clues regarding the specific traits responsible for differences in competitive ability between invasive annual and native perennial grasses on semiarid rangelands.

Soil morphology and organic matter dynamics under cheatgrass and sagebrush-steppe plant communities

Widespread cheatgrass (Bromus tectorum L.) invasion represents a major shift in species dominance that may alter ecosystem processes across much of the western US. To investigate differences following such conversion, soil morphology and organic matter under cheatgrass-dominated and native shrub-steppe vegetation were compared by standard soil analysis procedures at seven paired sites in Idaho and Utah. Results suggest that, following conversion to cheatgrass dominance, increased porosity and labile organic inputs enhance microbial decomposition in near-surface horizons beneath cheatgrass compared to adjacent soils under native vegetation. Enhanced decomposition could result in depletion of long-term SOM, leading to impoverished sites difficult to restore to native perennial vegetation.

Regulation and Measurement of Nitrification in Terrestrial Systems

Understanding nitrification rates and their regulation continues as a key area of research for assessing humans increasing impact on the terrestrial N cycle. We review the organisms and processes responsible for nitrification in terrestrial systems. The control of nitrification by substrate availability is discussed with particular attention to the factors affecting ammonia/ammonium availability. The effects on nitrification rates of environmental controls including oxygen, water potential, temperature and pH are described. With this general understanding of the factors affecting nitrification rates as a basis, we present an in depth analysis of methods used to measure nitrification in terrestrial systems. Net, gross and potential nitrification rate measurements are explained including the use of isotopes and inhibitors to measure rates in soils. Methods for the estimation of nitrification kinetics and modeling are briefly described. Future challenges will require understanding the factors controlling nitrification across spatial scales from ecosystems to soil microsites if we are to sustainably manage reactive nitrogen in terrestrial environments.

Enhanced removal of polychlorinated biphenyls from alfalfa rhizosphere soil in a field study: The impact of a rhizobial inoculum

Polychlorinated biphenyls (PCB) are persistent pollutants in soil environments where they continue to present considerable human health risks. Successful strategies to remediate contaminated soils are needed that are effective and of low cost. Bioremediation approaches that include the use of plants and microbial communities to promote degradation of PCB have significant potential but need further assessment under field conditions. The effects of growth of alfalfa (Medicago sativa L.) and inoculation with a symbiotic nitrogen fixing bacterium (Rhizobium meliloti) on the removal of polychlorinated biphenyls (PCB) from rhizosphere soil were evaluated in a field experiment. The initial PCB content of the soil ranged from 414 to 498 microg kg(-)(1). PCB removal for the rhizosphere soil was enhanced in the planted treatments, an average of 36% decrease in PCB levels compared to a 5.4% decrease in the unplanted soil, and further enhanced when plants were inoculated with the symbiotic Rhizobium (an average of 43% decrease) when evaluated at 90 days after planting. Plant biomass production was higher in the inoculated treatment. The total PCB content was increased from 3.30 microg kg(-)(1) to 26.72 microg kg(-)(1) in plant shoots, and from 115.07 microg kg(-)(1) to 142.23 microg kg(-)(1) in roots in the inoculated treatment compared to the planted treatment. Increased colony forming units (cfu) of total heterotrophic bacteria, biphenyl-degrading bacteria and fungi were observed in the rhizosphere of inoculated plants. PCB removal from the rhizosphere soil was not significantly correlated with the direct PCB uptake by the plants in any of the treatments but was significantly correlated with the stimulation of rhizosphere microflora. Changes in the soil microbial community structure in the planted and inoculated treatment were observed by profiling of bacterial ribosomal sequences. Some bacteria, such as Flavobacterium sp., may have contributed to the effective degradation of PCB and deserve further investigation.

Microbial control of nitrate concentrations in an agricultural soil treated with dairy waste compost or ammonium fertilizer

Abstract We conducted a 112-day laboratory incubation of an agricultural soil treated with dairy-waste compost or ammonium sulfate ((NH 4 ) 2 SO 4 ) to examine the role of microbial production and consumption of NO − 3 in controlling soil NO − 3 concentrations. Inorganic N, net N process rates, nitrification potentials and gross N process rates were measured at various time periods in the treated soils. Microbial consumption of NO − 3 was not an important process in controlling soil NO − 3 concentrations in these soil systems. Transient growth in the nitrifier population was observed with ammonium sulfate but not compost addition. Nitrification rates were significantly correlated with and comprised about 50% of the gross N mineralization rates, suggesting that nitrifying bacteria were not weaker competitors for soil NH + 4 than heterotrophs in these systems.