Nitrogen and Iron Availability Drive Metabolic Remodeling and Natural Selection of Diverse Phytoplankton during Experimental Upwelling.

Nearly half of carbon fixation and primary production originates from marine phytoplankton, and much of it occurs in episodic blooms in upwelling regimes. Here, we simulated blooms limited by nitrogen and iron by incubating Monterey Bay surface waters with subnutricline waters and inorganic nutrients and measured the whole-community transcriptomic response during mid- and late-bloom conditions. Cell counts revealed that centric and pennate diatoms (largely Pseudo-nitzschia and Chaetoceros spp.) were the major blooming taxa, but dinoflagellates, prasinophytes, and prymnesiophytes also increased. Viral mRNA significantly increased in late bloom and likely played a role in the bloom's demise. We observed conserved shifts in the genetic similarity of phytoplankton populations to cultivated strains, indicating adaptive population-level changes in community composition. Additionally, the density of single nucleotide variants (SNVs) declined in late-bloom samples for most taxa, indicating a loss of intraspecific diversity as a result of competition and a selective sweep of adaptive alleles. We noted differences between mid- and late-bloom metabolism and differential regulation of light-harvesting complexes (LHCs) under nutrient stress. While most LHCs are diminished under nutrient stress, we showed that diverse taxa upregulated specialized, energy-dissipating LHCs in low iron. We also suggest the relative expression of NRT2 compared to the expression of GSII as a marker of cellular nitrogen status and the relative expression of iron starvation-induced protein genes (ISIP1, ISIP2, and ISIP3) compared to the expression of the thiamine biosynthesis gene (thiC) as a marker of iron status in natural diatom communities. IMPORTANCE Iron and nitrogen are the nutrients that most commonly limit phytoplankton growth in the world's oceans. The utilization of these resources by phytoplankton sets the biomass available to marine systems and is of particular interest in high-nutrient, low-chlorophyll (HNLC) coastal fisheries. Previous research has described the biogeography of phytoplankton in HNLC regions and the transcriptional responses of representative taxa to nutrient limitation. However, the differential transcriptional responses of whole phytoplankton communities to iron and nitrogen limitation has not been previously described, nor has the selective pressure that these competitive bloom environments exert on major players. In addition to describing changes in the physiology of diverse phytoplankton, we suggest practical indicators of cellular nitrogen and iron status for future monitoring.

The utility of functional gene arrays for assessing community composition, relative abundance, and distribution of ammonia-oxidizing bacteria and archaea.

Ammonia-oxidizing bacteria (AOB) and archaea (AOA) transform ammonium to nitrite, an essential step in the complete mineralization of organic matter, leading to the accumulation of nitrate in oxic environments. The diversity and community composition of both groups have been extensively explored by sequence analysis of both 16S rRNA and amoA (encoding the critical enzyme, ammonia monooxygenase subunit A) genes. In this chapter, the power of the amoA gene as a phylogenetic marker for both AOB and AOA is extended to the development and application of DNA microarrays. Functional gene microarrays provide high throughput, relatively high resolution data on community composition and relative abundance, which is especially useful for comparisons among environments, and between samples in time and space, targeting the microbial group that is responsible for a biogeochemical transformation of interest, such as nitrification. In this chapter, the basic approaches to the design of probes to represent the target groups AOB and AOA are described, and the protocols for preparing hybridization targets from environmental samples are provided. Factors that influence the hybridization results and determine the sensitivity and specificity of the assays are discussed. A few examples of recent applications of amoA microarrays to explore temporal and spatial patterns in AOB and AOA community composition in estuaries and the ocean are presented. Array data are lower resolution than sequencing, but much higher throughput, thus allowing robust statistics and reproducibility that are not possible with large clone libraries. For specific functional groups, arrays provide more direct information in a more economical format than is possible with next generation sequencing.

Measurement and distribution of nitrification rates in the oceans.

Nitrification is the process that converts ammonium to nitrate and thus links the regeneration of organic nitrogen to fixed nitrogen loss by denitrification. The first step, oxidation of ammonia to nitrite, is performed by a phylogenetically restricted group of proteobacteria (ammonia-oxidizing bacteria, AOB) and Crenarchaea (ammonia-oxidizing archaea, AOA). The second step is restricted to nitrite-oxidizing bacteria (NOB) as far as currently known. All three groups are assumed to be autotrophic and obligately aerobic, but the true extent of autotrophy and potential anaerobic pathways in these organisms is currently under investigation. Here, we describe methods for the measurement of nitrification rates in the marine environment, with a focus on seawater systems and stable isotopic tracer methods. The methods vary in analytical requirements but share the need for incubations, which must be optimized for different environments with different substrate concentrations. Recent advances in mass spectrometry now make it possible to minimize incubation artifacts and to achieve greatly improved sensitivity.

Bioengineering strategies for regeneration of craniofacial bone: a review of emerging technologies.

Although advances in surgical techniques and bone grafting have significantly improved the functional and cosmetic restoration of craniofacial structures lost because of trauma or disease, there are still significant limitations in our ability to regenerate these tissues. The regeneration of oral and craniofacial tissues presents a formidable challenge that requires synthesis of basic science, clinical science, and engineering technology. Tissue engineering is an interdisciplinary field of study that addresses this challenge by applying the principles of engineering to biology and medicine toward the development of biological substitutes that restore, maintain, and improve normal function. This review will explore the impact of biomaterials design, stem cell biology and gene therapy on craniofacial tissue engineering.

Bone regeneration in defects compromised by radiotherapy.

Because bone reconstruction in irradiated sites is less than ideal, we applied a regenerative gene therapy method in which a cell-signaling virus was localized to biomaterial scaffolds to regenerate wounds compromised by radiation therapy. Critical-sized defects were created in rat calvariae previously treated with radiation. Gelatin scaffolds containing lyophilized adenovirus encoding BMP-2 (AdBMP-2) or freely suspended AdBMP-2 were transplanted. Lyophilized AdBMP-2 significantly improved bone quality and quantity over free AdBMP-2. Bone mineral density was reduced after radiotherapy. Histological analyses demonstrated that radiation damage led to less bone regeneration. The woven bone and immature marrow formed in the radiated defects indicated that irradiation retarded normal bone development. Finally, we stored the scaffolds with lyophilized AdBMP-2 at -80 degrees C to determine adenovirus stability. Micro-CT quantification demonstrated no significant differences between bone regeneration treated with lyophilized AdBMP-2 before and after storage, suggesting that virus-loaded scaffolds may be convenient for application as pre-made constructs.

Denitrification as the dominant nitrogen loss process in the Arabian Sea.

Primary production in over half of the world's oceans is limited by fixed nitrogen availability. The main loss term from the fixed nitrogen inventory is the production of dinitrogen gas (N(2)) by heterotrophic denitrification or the more recently discovered autotrophic process, anaerobic ammonia oxidation (anammox). Oceanic oxygen minimum zones (OMZ) are responsible for about 35% of oceanic N(2) production and up to half of that occurs in the Arabian Sea. Although denitrification was long thought to be the only loss term, it has recently been argued that anammox alone is responsible for fixed nitrogen loss in the OMZs. Here we measure denitrification and anammox rates and quantify the abundance of denitrifying and anammox bacteria in the OMZ regions of the Eastern Tropical South Pacific and the Arabian Sea. We find that denitrification rather than anammox dominates the N(2) loss term in the Arabian Sea, the largest and most intense OMZ in the world ocean. In seven of eight experiments in the Arabian Sea denitrification is responsible for 87-99% of the total N(2) production. The dominance of denitrification is reproducible using two independent isotope incubation methods. In contrast, anammox is dominant in the Eastern Tropical South Pacific OMZ, as detected using one of the isotope incubation methods, as previously reported. The abundance of denitrifying bacteria always exceeded that of anammox bacteria by up to 7- and 19-fold in the Eastern Tropical South Pacific and Arabian Sea, respectively. Geographic and temporal variability in carbon supply may be responsible for the different contributions of denitrification and anammox in these two OMZs. The large contribution of denitrification to N(2) loss in the Arabian Sea indicates the global significance of denitrification to the oceanic nitrogen budget.

Denitrifying bacterial community composition changes associated with stages of denitrification in oxygen minimum zones.

Denitrification in the ocean is a major sink for fixed nitrogen in the global N budget, but the process is geographically restricted to a few oceanic regions, including three oceanic oxygen minimum zones (OMZ) and hemipelagic sediments worldwide. Here, we describe the diversity and community composition of microbes responsible for denitrification in the OMZ using polymerase chain reaction, sequence and fragment analysis of clone libraries of the signature genes (nirK and nirS) that encode the enzyme nitrite reductase, responsible for key denitrification transformation steps. We show that denitrifying assemblages vary in space and time and exhibit striking changes in diversity associated with the progression of denitrification from initial anoxia through nitrate depletion. The initial denitrifying assemblage is highly diverse, but succession on the scale of 3-12 days leads to a much less diverse assemblage and dominance by one or a few phylotypes. This progression occurs in the natural environment as well as in enclosed incubations. The emergence of dominants from a vast reservoir of rare types has implications for the maintenance of diversity of the microbial population and suggests that a small number of microbial dominants may be responsible for the greatest rates of transformations involving nitrous oxide and global fixed nitrogen loss. Denitrifying blooms, driven by a few types responding to episodic environmental changes and distributed unevenly in time and space, are consistent with the sampling effect model of diversity-function relationships. Canonical denitrification thus appears to have important parallels with both primary production and nitrogen fixation, which are typically dominated by regionally and temporally restricted blooms that account for a disproportionate share of these processes worldwide.

PHYTOPLANKTON COMMUNITY COMPOSITION AND GENE EXPRESSION OF FUNCTIONAL GENES INVOLVED IN CARBON AND NITROGEN ASSIMILATION(1).

A functional gene microarray was developed and used to investigate phytoplankton community composition and gene expression in the English Channel. Genes encoding the CO2 -fixation enzyme RUBISCO (rbcL) and the nitrate assimilation enzyme nitrate reductase (NR) representing several major groups of phytoplankton were included as oligonucleotide probes on the "phytoarray." Five major groups of eukaryotic phytoplankton that possess the Type 1D rbcL gene were detected, both in terms of presence (DNA) and activity (rbcL gene expression). Changes in relative signal intensity among the Type 1D rbcL probes indicated a shift from diatom dominance in the spring bloom to dominance by haptophytes and flagellates later in the summer. Because of the limitations of a smaller database, NR probes detected fewer groups, but due to the greater diversity among known NR sequences, NR probes provided higher phylogenetic resolution than did rbcL probes and identified two uncultivated diatom phylotypes as the most abundant (DNA) and active (NR gene expression) in field samples. Unidentified chlorophytes and the diatom Phaeodactylum tricornutum Bohlin were detected at both the DNA and cDNA (gene expression) levels. The reproducibility of the array was evaluated in several ways, and future directions for further improvement of probe development and sensitivity are outlined. The phytoarray provides a relatively high-resolution, high-throughput approach to assessing phytoplankton community composition in marine environments.

Relationship of temporal and spatial variabilities of ammonia-oxidizing bacteria to nitrification rates in Monterey Bay, California.

Temporal and spatial dynamics of ammonia-oxidizing bacteria (AOB) were examined using genes encoding 16S rRNA and ammonia monooxygenase subunit A (AmoA) in Monterey Bay, Calif. Samples were collected from three depths in the water column on four dates at one mid-bay station. Diversity estimators for the two genes showed a strong positive correlation, indicating that overlapping bacterial populations had been sampled by both sets of clone libraries. Some samples that were separated by only 15 m in depth had less genetic similarity than samples that were collected from the same depth months apart. Clone libraries from the Monterey Bay AOB community were dominated by Nitrosospira-like sequences and clearly differentiated from the adjacent AOB community in Elkhorn Slough. Many Monterey Bay clones clustered with previously identified 16S rRNA and amoA groups composed entirely of marine sequences, supporting the hypothesis that these groups are specific to the marine environment and are dominant marine AOB. In addition, novel, phylogenetically distinct groups of AOB sequences were identified and compared to sequences in the database. Only one cluster of gammaproteobacterial AOB was detected using 16S rRNA genes. Although significant genetic variation was detected in AOB populations from both vertical and temporal samples, no significant correlation was detected between diversity and environmental variables or the rate of nitrification.

Dissimilatory nitrite reductase genes from autotrophic ammonia-oxidizing bacteria.

The presence of a copper-containing dissimilatory nitrite reductase gene (nirK) was discovered in several isolates of beta-subdivision ammonia-oxidizing bacteria using PCR and DNA sequencing. PCR primers Cunir3 and Cunir4 were designed based on published nirK sequences from denitrifying bacteria and used to amplify a 540-bp fragment of the nirK gene from Nitrosomonas marina and five additional isolates of ammonia-oxidizing bacteria. Amplification products of the expected size were cloned and sequenced. Alignment of the nucleic acid and deduced amino acid (AA) sequences shows significant similarity (62 to 75% DNA, 58 to 76% AA) between nitrite reductases present in these nitrifiers and the copper-containing nitrite reductase found in classic heterotrophic denitrifiers. While the presence of a nitrite reductase in Nitrosomonas europaea is known from early biochemical work, preliminary sequence data from its genome indicate a rather low similarity to the denitrifier nirKs. Phylogenetic analysis of the partial nitrifier nirK sequences indicates that the topology of the nirK tree corresponds to the 16S rRNA and amoA trees. While the role of nitrite reduction in the metabolism of nitrifying bacteria is still uncertain, these data show that the nirK gene is present in closely related nitrifying isolates from many oceanographic regions and suggest that nirK sequences retrieved from the environment may include sequences from ammonia-oxidizing bacteria.

Vertical Distribution of Denitrification Potential, Denitrifying Bacteria, and Benzoate Utilization in Intertidal Microbial Mat Communities.

Aspects of denitrification and benzoate degradation were studied in two estuarine microbial mat communities on the California coast by measuring the depth distributions of potential denitrification rates, genetic potential for denitrification, nitrate concentration, benzoate mineralization rates, total bacterial abundance, and abundance of a denitrifying strain (TBD-8b) isolated from one of the sites. Potential denitrification was detected in microbial mat cores from both Elkhorn Slough and Tomales Bay. Maximum denitrification rates were more than two orders of magnitude higher at Elkhorn Slough (3.14 mmol N m -2 d -1) than at Tomales Bay (0.02 mmol N m -2 d -1), and at both sites, the maximum rates occurred in the 0-2 mm depth interval. Ambient pore [NO 3 + NO 2] was substantially higher at Elkhorn Slough than at Tomales Bay. Incorporation and mineralization of benzoate was maximal near the mat surface at Elkhorn Slough. The areal rate of benzoate utilization was 1045 nmol C m -2 d -1, which represented utilization of 70% of the added substrate in 24 h. Total bacterial and TBD-8b abundances were greatest near the surface at both Tomales Bay and Elkhorn Slough, and TBD-8b represented less than 0.2% of the total. Genetic potential for denitrification, quantified by hybridization with a nitrite reductase gene fragment, was present below the mat surface at average levels representing presence of the gene in approximately 10% of the total cells.

Molecular analysis of ammonia oxidation and denitrification in natural environments.

This review summarizes aspects of the current knowledge about the ecology of ammonia-oxidizing and denitrifying bacteria. The development of molecular techniques has contributed enormously to the rapid recent progress in the field. Different techniques for doing so are discussed. The characterization of ammonia-oxidizing and -denitrifying bacteria by sequencing the genes encoding 16S rRNA and functional proteins opened the possibility of constructing specific probes. It is now possible to monitor the occurrence of a particular species of these bacteria in any habitat and to get an estimate of the relative abundance of different types, even if they are not culturable as yet. These data indicate that the composition of nitrifying and denitrifying communities is complex and apparently subject to large fluctuations, both in time and in space. More attempts are needed to enrich and isolate those bacteria which dominate the processes, and to characterize them by a combination of physiological, biochemical and molecular techniques. While PCR and probing with nucleotides or antibodies are primarily used to study the structure of nitrifying and denitrifying communities, studies of their function in natural habitats, which require quantification at the transcriptional level, are currently not possible.

Analysis of ammonia-oxidizing bacteria from hypersaline Mono Lake, California, on the basis of 16S rRNA sequences.

Ammonia-oxidizing bacteria were detected by PCR amplification of DNA extracted from filtered water samples throughout the water column of Mono Lake, California. Ammonia-oxidizing members of the beta subdivision of the division Proteobacteria (beta-subdivision Proteobacteria) were detected using previously characterized PCR primers; target sequences were detected by direct amplification in both surface water and below the chemocline. Denaturing gradient gel electrophoresis analysis indicated the presence of at least four different beta-subdivision ammonia oxidizers in some samples. Subsequent sequencing of amplified 16S rDNA fragments verified the presence of sequences very similar to those of cultured Nitrosomonas strains. Two separate analyses, carried out under different conditions (different reagents, locations, PCR machines, sequencers, etc.), 2 years apart, detected similar ranges of sequence diversity in these samples. It seems likely that the physiological diversity of nitrifiers exceeds the diversity of their ribosomal sequences and that these sequences represent members of the Nitrosomonas europaea group that are acclimated to alkaline, high-salinity environments. Primers specific for Nitrosococcus oceanus, a marine ammonia-oxidizing bacterium in the gamma subdivision of the Proteobacteria, did not amplify target from any samples.

Nitrification and ammonification in aquatic systems.

Nitrification is an essential step in the nitrogen cycle of natural systems because it links organic matter degradation to fixed nitrogen loss. Ammonium released by ammonification is oxidized to nitrate by nitrification, and can then be reduced to dinitrogen gas by denitrification, resulting in net loss of fixed nitrogen from the system. Whether organic matter degradation results in net ammonium release depends largely on the quality of the organic substrate and interactions among members of the microbial community involved in nitrogen and organic matter cycling. In sediments, nitrogen cycle processes depend on the supply of organic matter and oxygen from overlying water. The nature of the net flux (which direction and which form of nitrogen) is a function of closely coupled reactions (ammonification-nitrification-denitrification) in the nitrogen cycle.

Detection of ammonium-oxidizing bacteria of the beta-subclass of the class Proteobacteria in aquatic samples with the PCR.

The PCR was used as the basis for the development of a sensitive and specific assay for the detection of ammonium-oxidizing bacteria belonging to the beta-subclass of the class Proteobacteria. PCR primers were selected on the basis of nucleic acid sequence data available for seven species of nitrifiers in this subclass. The specificity of the ammonium oxidizer primers was evaluated by testing known strains of nitrifiers, several serotyped environmental nitrifier isolates, and other members of the Proteobacteria, including four very closely related, nonnitrifying species (as determined by rRNA sequence analysis). DNA extracts from 19 bacterio-plankton samples collected from Lake Bonney, Antarctica, and the Southern California Bight were assayed for the presence of ammonium oxidizers. By using a two-stage amplification procedure, ammonium oxidizers were detected in samples collected from both sites. Chemical data collected simultaneously support the occurrence of nitrification and the presence of nitrifiers. This is the first report describing PCR primers specific for ammonium-oxidizing bacteria and the successful amplification of nitrifier genes coding for rRNA from DNA extracts from natural samples. This application of PCR is of particular importance for the detection and study of microbes, such as autotrophic nitrifiers, which are difficult or impossible to isolate from indigenous microbial communities.

Nitrogen uptake, dissolved organic nitrogen release, and new production.

In oceanic, coastal, and estuarine environments, an average of 25 to 41 percent of the dissolved inorganic nitrogen (NH(4) (+) and NO(3) (-)) taken up by phytoplankton is released as dissolved organic nitrogen (DON). Release rates for DON in oceanic systems range from 4 to 26 nanogram-atoms of nitrogen per liter per hour. Failure to account for the production of DON during nitrogen-15 uptake experiments results in an underestimate of gross nitrogen uptake rates and thus an underestimate of new and regenerated production. In these studies, traditional nitrogen-15 techniques were found to underestimate new and regenerated production by up to 74 and 50 percent, respectively. Total DON turnover times, estimated from DON release resulting from both NH(4) (+) and NO(3) (-) uptake, were 10 +/- 1, 18 +/- 14, and 4 days for oceanic, coastal, and estuarine sites, respectively.

Antibody and DNA probes for detection of nitrite reductase in seawater.

A polyclonal antiserum was produced by immunization with nitrite reductase (NiR) purified from Pseudomonas stutzeri (ATCC 14405) and tested for specificity among known denitrifying strains. The antiserum was nearly strain-specific, identifying NiR only in some, but not all, other P. stutzeri strains. Denitrifying isolates from water column and sediment environments were also screened; several isolates from an intertidal microbial mat reacted with the NiR antiserum. Activity assays for NiR in polyacrylamide gels demonstrated that strains with apparently very similar NiR proteins did not react with the antiserum. These results imply that the NiR protein is more variable even among closely related strains than previously suspected. A DNA probe for a 721 bp region of the NiR structural gene was obtained by PCR amplification of P. stutzeri (ATCC 14405) DNA and used to screen denitrifying strains and isolates. The probe hybridized with a greater variety of strains than did the antiserum, implying that the DNA probe may be a more broadly useful and functional probe in environmental samples, whilst the NiR antiserum is nearly strain- or, at most, species-specific. Limits for detection of the enzyme and gene in seawater were estimated and NiR DNA was detected in DNA extracted from natural seawater. The hybridization data imply that in the order of 1-10 in 1000 cells in natural seawater possess homology with the NiR gene probe.

Selective inhibition of ammonium oxidation and nitrification-linked n(2)o formation by methyl fluoride and dimethyl ether.

Methyl fluoride (CH(3)F) and dimethyl ether (DME) inhibited nitrification in washed-cell suspensions of Nitrosomonas europaea and in a variety of oxygenated soils and sediments. Headspace additions of CH(3)F (10% [vol/vol]) and DME (25% [vol/vol]) fully inhibited NO(2) and N(2)O production from NH(4) in incubations of N. europaea, while lower concentrations of these gases resulted in partial inhibition. Oxidation of hydroxylamine (NH(2)OH) by N. europaea and oxidation of NO(2) by a Nitrobacter sp. were unaffected by CH(3)F or DME. In nitrifying soils, CH(3)F and DME inhibited N(2)O production. In field experiments with surface flux chambers and intact cores, CH(3)F reduced the release of N(2)O from soils to the atmosphere by 20- to 30-fold. Inhibition by CH(3)F also resulted in decreased NO(3) + NO(2) levels and increased NH(4) levels in soils. CH(3)F did not affect patterns of dissimilatory nitrate reduction to ammonia in cell suspensions of a nitrate-respiring bacterium, nor did it affect N(2)O metabolism in denitrifying soils. CH(3)F and DME will be useful in discriminating N(2)O production via nitrification and denitrification when both processes occur and in decoupling these processes by blocking NO(2) and NO(3) production.

Comparison of Nucleic Acid Hybridization and Fluorometry for Measurement of the Relationship between RNA/DNA Ratio and Growth Rate in a Marine Bacterium.

Continuous culture of Pseudomonas stutzeri Zobell, a marine denitrifying bacterium, was used to determine the relationship between growth rate and nucleic acid content. The trend of decreasing RNA content with decreasing growth rate, well known for enteric organisms, was found to occur in P. stutzeri Zobell as well, even at very long generation times such as those thought to occur in the oligotrophic ocean. When assayed by ethidium bromide fluorescence, the total RNA/DNA ratio was linear for generation times between 6 and 60 h. We also developed a 200-bp nucleic acid probe (with species-specific potential) for a portion of the 23S rRNA gene of P. stutzeri Zobell, which was used to quantify rRNA and rDNA by hybridization in the same continuous cultures. The rRNA/rDNA ratio also exhibited a decrease with decreasing growth rate, but the relationship, although significant, was not simply linear. The sensitivity and accuracy of the two methods are compared, and the potential for species specificity in future hybridizations is discussed.