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Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity.

ABSTRACT: A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml(-1), a half-saturation of signal at 0.26 ng ml(-1), and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.

SUBMITTER: Steward GF

PROVIDER: S-EPMC368376 | biostudies-literature | 2004 Mar

REPOSITORIES: biostudies-literature

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Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity.

Steward Grieg F GF Jenkins Bethany D BD Ward Bess B BB Zehr Jonathan P JP

Applied and environmental microbiology 20040301 3

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signa ...[more]

PMID: 15006766

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Project description:BackgroundHolobionts comprising nitrogen-fixing diazotrophs and phytoplankton or zooplankton are ubiquitous in the pelagic sea. However, neither the community structure of plankton-associated diazotrophs (PADs) nor their nitrogenase transcriptional activity are well-understood. In this study, we used nifH gene Illumina sequencing and quantitative PCR to characterize the community composition and nifH expression profile of PADs with > 100 μm size fraction in the euphotic zone of the northern South China Sea.ResultsThe results of DNA- and RNA-derived nifH gene revealed a higher alpha-diversity in the active than in the total community. Moreover, the compositional resemblance among different sites was less for active than for total communities of PADs. We characterized the 20 most abundant OTUs by ranking the sum of sequence reads across 9 sampling stations for individual OTUs in both nifH DNA and RNA libraries, and then assessed their phylogenetic relatedness. Eight of the 20 abundant OTUs were phylogenetically affiliated with Trichodesmium and occurred in approximately equal proportion in both the DNA and RNA libraries. The analysis of nifH gene expression level showed uneven attribute of the abundance and nitrogenase activities by the remaining 12 OTUs. Taxa belonging to cluster III and Betaproteobacteria were present at moderate abundance but exhibited negligible nitrogenase transcription activity. Whereas, the abundances of Richelia, Deltaproteobacteria and Gammaproteobacteria were low but the contribution of these groups to nitrogenase transcription was disproportionately high.ConclusionsThe substantial variation in community structure among active dizatrophic fractions compared to the total communities suggests that the former are better indicators of biological response to environmental changes. Altogether, our study highlights the importance of rare PADs groups in nitrogen fixation in plankton holobionts, evidenced by their high level of nitrogenase transcription.

Project description:The Tropical North Atlantic (TNAtl) plays a critical role in the marine nitrogen cycle, as it supports high rates of biological nitrogen (N(2)) fixation, yet it is unclear whether this process is limited by the availability of iron (Fe), phosphate (P) or is co-limited by both. In order to investigate the impact of nutrient limitation on the N(2)-fixing microorganisms (diazotrophs) in the TNAtl, trace metal clean nutrient amendment experiments were conducted, and the expression of nitrogenase (nifH) in cyanobacterial diazotrophs in response to the addition of Fe, P, or Fe+P was measured using quantitative PCR. To provide context, N(2) fixation rates associated with the <10 ?m community and diel nifH expression in natural cyanobacterial populations were measured. In the western TNAtl, nifH expression in Crocosphaera, Trichodesmium, and Richelia was stimulated by Fe and Fe+P additions, but not by P, implying that diazotrophs may be Fe-limited in this region. In the eastern TNAtl, nifH expression in unicellular cyanobacteria UCYN-A and Crocosphaera was stimulated by P, implying P-limitation. In equatorial waters, nifH expression in Trichodesmium was highest in Fe+P treatments, implying co-limitation in this region. Nutrient additions did not measurably stimulate N(2) fixation rates in the <10 ?m fraction in most of the experiments, even when upregulation of nifH expression was evident. These results demonstrate the utility of using gene expression to investigate the physiological state of natural populations of microorganisms, while underscoring the complexity of nutrient limitation on diazotrophy, and providing evidence that diazotroph populations are slow to respond to the addition of limiting nutrients and may be limited by different nutrients on basin-wide spatial scales. This has important implications for our current understanding of controls on N(2) fixation in the TNAtl and may partially explain why it appears to be intermittently limited by Fe, P, or both.

Project description:DNA arrays are useful tools for simultaneously studying the expressions of a large number of genes. Herein, we describe the construction and the optimization of conditions for a low-density DNA macroarray specific for the porcine immune system. This specific DNA macroarray contains 63 gene products, including 20 cytokines, 11 chemokines, and 12 immunologically relevant receptors. It was constructed by designing gene-specific oligonucleotide primers from porcine sequences available in the EMBL or TIGR expressed sequence tag data bank and using primers from conserved regions of aligned sequences from other species for sequences unavailable for swine. Amplicons produced by reverse transcription-PCR were cloned, sequenced, and spotted onto nylon filters. A trial DNA array was first produced to optimize the intensity, specificity, and variability of signals from amplicons amplified with either gene-specific or universal primers. The DNA macroarray was then validated by comparing the gene expression profile of nonstimulated peripheral blood mononuclear cells (PBMCs) to that of phorbol 12-myristate 13-acetate and ionomycin (PMA-Iono)-stimulated PBMCs from three different animals over a 48-h time period. As already described for more conventional techniques, we showed that certain genes, such as those for CD40, gamma interferon, interleukin 2 (IL-2), the IL-2 receptor, and tumor necrosis factor alpha, were upregulated in PMA-Iono-stimulated PBMCs. A detailed analysis also indicated a downregulation of several genes which are expressed mainly by macrophages (IL-1, IL-8, AMCF-1, natural-resistance-associated macrophage protein, neutrophil chemotactic protein, DAP-12, and monocyte chemoattractant protein) in samples stimulated for 24 h with PMA-Iono compared to their levels of expression in control samples. These results indicate that the DNA macroarray that we constructed can be a useful tool for simultaneously monitoring the mRNA expression of immunologically relevant genes in different porcine samples.

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Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity.

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Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity.

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