Atlas of Signaling for Interpretation of Microarray Experiments

Microarray-based expression profiling of living systems is a quick and inexpensive method to obtain insights into the nature of various diseases and phenotypes. A typical microarray profile can yield hundreds or even thousands of differentially expressed genes and finding biologically plausible themes or regulatory mechanisms underlying these changes is a non-trivial and daunting task. We describe a novel approach for systems-level interpretation of microarray expression data using a manually constructed “overview” pathway depicting the main cellular signaling channels (Atlas of Signaling). Currently, the developed pathway focuses on signal transduction from surface receptors to transcription factors and further transcriptional regulation of cellular “workhorse” proteins. We show how the constructed Atlas of Signaling in combination with an enrichment analysis algorithm allows quick identification and visualization of the main signaling cascades and cellular processes affected in a gene expression profiling experiment. We validate our approach using several publicly available gene expression datasets

Lake Superior Phototrophic Picoplankton: Nitrate Assimilation Measured with a Cyanobacterial Nitrate-responsive Bioreporter and Genetic Diversity of the Natural Community

Cyanobacteria of the picoplankton size range (picocyanobacteria) Synechococcus and Prochlorococcus contribute significantly to total phytoplankton biomass and primary production in marine and freshwater oligotrophic environments. Despite their importance, little is known about the biodiversity and physiology of freshwater picocyanobacteria. Lake Superior is an ultra-oligotrophic system with light and temperature conditions unfavorable for photosynthesis. Synechococcus-like picocyanobacteria are an important component of phytoplankton in Lake Superior. The concentration of nitrate, the major form of combined nitrogen in the lake, has been increasing continuously in these waters over the last 100 years, while other nutrients remained largely unchanged. Decreased biological demand for nitrate caused by low availabilities of phosphorus and iron, as well as low light and temperature was hypothesized to be one of the reasons for the nitrate build-up. One way to get insight into the microbiological processes that contribute to the accumulation of nitrate in this ecosystem is to employ a cyanobacterial bioreporter capable of assessing the nitrate assimilation capacity of phytoplankton. In this study, a nitrate-responsive biorepoter AND100 was constructed by fusing the promoter of the Synechocystis PCC 6803 nitrate responsive gene nirA, encoding nitrite reductase to the Vibrio fischeri luxAB genes, which encode the bacterial luciferase, and genetically transforming the resulting construct into Synechocystis. The transcription of luciferase in the transformant is regulated by the availability of nitrate in the sample. Therefore, the bioluminescent signal produced by the bioreporter reflects the nitrate assimilation capacity of the cell. The dynamic range of the bioreporter response was found to be between 1 and 100 µM nitrate. The results of a series of bioreporter assays conducted on preserved water samples collected from several stations in Lake Superior in May and September 2004 suggest that low availability of phosphorus is the major factor that constrains nitrate depletion in the lake with low seasonal or spatial variability. In addition, iron was found to be a secondary limiting factor, whose effect is evident only of phosphorus is added to the sample. During the period of isothermal mixing, light was shown to significantly reduce nitrate depletion in the lake. Overall, the bioreporter AND100 is a suitable model for elucidating the factors that regulate nitrate depletion by phytoplankton in natural waters. However, understanding the physiology of the natural cyanobacterial assemblages in the lake helps to prove the validity of the bioreporter approach. Since the information on the endemic Lake Superior phytoplankton is very scarce, an initial characterization of the genetic diversity of cyanobacteria in the lake was conducted. High throughput sequencing of a library of cyanobacterial 16S ribosomal DNA clones amplified by PCR from DNA isolated from the lake water resulted in 368 successful reactions. In a neighbor-joining tree the majority of the 16S rDNA sequences clustered within the “picocyanobacterial clade” that consists of both freshwater and marine Synechococcus and Prochlorococcus picocyanobacteria. Two new groups of picocyanobacteria LSI and II that do not cluster within any of the known freshwater picocyanobacterial clusters were the most abundant (\u3e 50% of the sequences) in the samples collected from pelagic Lake Superior stations. Conversely, at station KW located in a nearshore urban area, only 4% of the sequences belonged to these clusters, and the remaining of the sequences reflected the freshwater biodiversity described previously. In addition, several picocyanobacterial strains were isolated from Lake Superior between years 2004 and 2005. Despite their low representation in the ..

Construction and characterization of a cyanobacterial bioreporter capable of assessing nitrate assimilatory capacity in freshwaters

The use of cyanobacterial whole-cell luminescent bioreporters has enhanced our ability to monitor nutrient availability in aquatic ecosystems. We have constructed a Synechocystis sp. strain PCC6803 bioluminescent reporter for the assessment of nitrate bioavailability. Specifically, a 380-base pair DNA fragment containing the NtcA/B-dependent nitrate/nitrite-activated nirA promoter (regulating expression of genes encoding nitrite reductase) was fused to the bacterial luciferase genes, luxAB, and introduced into Synechocystis by genetic transformation. Characterization of this strain, designated AND100, yielded dose-dependent increased bioluminescence coincident with increased nitrate added to the growth medium from 1 to 100 μM. Bioluminescence in response to nitrate addition was light dependent up to 50 μmol quanta m-2 s-1. Assessing environmental samples collected from oligotrophic Lake Superior, we demonstrated that the onset of luminescence coincided with the drawdown of nitrate by simultaneously monitoring nitrate depletion from reaction vessels. Nitrate in the Lake Superior samples was consistently underestimated by the bioreporter. Only by following amendment of these samples with phosphate and iron was total nitrate accurately reflected by the cyanobacterial bioreporter. Thus, strain AND 100 can be used to elucidate factors that constrain use of nitrate in freshwaters. This is pertinent to a system such as Lake Superior where the concentration of nitrate has increased 6-fold in the last century. Indeed, pilot experiments with the bioreporter suggest that nutrient co-limitation (P and Fe), as well as low light, may reduce the capacity for nitrate assimilation in field samples from Lake Superior. © 2005, by the American Society of Limnology and oceanography, Inc

CYANOBACTERIA OF THE GENUS PROCHLOROTHRIX

Green cyanobacteria are distinguished from blue-green ones by the possession of a chlorophyll-containing light harvesting antenna. Three genera of green cyanobacteria, namely Acaryochloris, Prochlorococcus and Prochloron, are unicellular and of marine habitat; Prochlorococcus marinus attracts most attention due to its outstanding role in prime productivity. The fourth genus, Prochlorothrix, is represented by filamentous freshwater strains. Unlike the rest of green cyanobacteria, Prochlorothrix is paradoxically rare: it has been isolated from two European locations only. Taking into account fluctuating blooms, morphological resemblance with Planktothrix and Pseudanabaena, and unsuccessful enrichment of Prochlorothrix, the preferred strategy of search for this cyanobacterium is based on PCR with natural DNA and specific primers. This approach already demonstrates a broader distribution of Prochlorothrix: marker genes have been found in at least two additional locations. Despite the growing evidence for naturally occurring Prochlorothrix, there are only a few cultivated strains, and only one of them (PCC 9006) is claimed to be axenic. In multixenic cultures, Prochlorothrix is accompanied by heterotrophic bacteria, indicating a consortium-type association. The genus Prochlorothrix includes two species: P. hollandica and P. scandica based on distinctions in genomic DNA, cell size, temperature optimum, and fatty acid composition of membrane lipids. In this short review, the properties of cyanobacteria of the genus Prochlorothrix are described, and the evolutionary scenario of green cyanobacteria, especially taking into account their role in the origin of simple chloroplast is given

Nitrate utilization by phytoplankton in Lake Superior is impaired by low nutrient (P, Fe) availability and seasonal light limitation - A cyanobacterial bioreporter study

We previously developed a luminescent Synechococystis sp. strain PCC 6803 cyanobacterial bioreporter that is used as a real-time whole-cell sensor to assess nitrate assimilatory capacity in freshwaters. Applying the bioreporter assay to Lake Superior, a system whose nitrate levels have increased 6-fold since 1900, we investigated factors that constrain nitrate utilization in this oligotrophic system. Clean sampling methods were used to collect water from Lake Superior during spring and summer 2004, and nitrate utilization was measured by monitoring bioreporter luminescence. Bioreporter response was monitored during experiments in which the lake water was amended with nutrients and incubated under light regimes simulating integrated spring and summer mixing depths. These studies demonstrated that nitrate utilization was enhanced at most stations following addition of phosphorus (P). Moreover, at many stations, addition of iron (Fe) enhanced the P effect. Strength-of-effect statistical analysis provided the individual contribution of P and Fe toward stimulating bioreporter response. In general, distance from shore and season were not good predictors of nitrate assimilatory capacity. Manipulation of light flux during bioreporter experiments also showed that light intensities experienced during spring mixing are likely insufficient to saturate the rate of nitrate utilization. Overall, these data suggest that P-limited algae are deficient in their ability to assimilate nitrate in Lake Superior. Furthermore, we suggest that a secondary limitation for Fe may occur that further constrains nitrate drawdown. Lastly, during spring, light fluxes are sufficiently low to prevent maximal nitrate utilization, even in the absence of nutrient limitation. © 2007 Phycological Society of America

Lake Superior Supports Novel Clusters of Cyanobacterial Picoplankton▿ †

Very little is known about the biodiversity of freshwater autotrophic picoplankton (APP) in the Laurentian Great Lakes, a system comprising 20% of the world's lacustrine freshwater. In this study, the genetic diversity of Lake Superior APP was examined by analyzing 16S rRNA gene and cpcBA PCR amplicons from water samples. By neighbor joining, the majority of 16S rRNA gene sequences clustered within the “picocyanobacterial clade” consisting of freshwater and marine Synechococcus and Prochlorococcus. Two new groups of Synechococcus spp., the pelagic Lake Superior clusters I and II, do not group with any of the known freshwater picocyanobacterial clusters and were the most abundant species (50 to 90% of the sequences) in samples collected from offshore Lake Superior stations. Conversely, at station Portage Deep (PD), located in a nearshore urbanized area, only 4% of the sequences belonged to these clusters and the remaining clones reflected the freshwater Synechococcus diversity described previously at sites throughout the world. Supporting the 16S rRNA gene data, the cpcBA library from nearshore station PD revealed a cosmopolitan diversity, whereas the majority of the cpcBA sequences (97.6%) from pelagic station CD1 fell within a unique Lake Superior cluster. Thus far, these picocyanobacteria have not been cultured, although their phylogenetic assignment suggests that they are phycoerythrin (PE) rich, consistent with the observation that PE-rich APP dominate Lake Superior picoplankton. Lastly, flow cytometry revealed that the summertime APP can exceed 105 cells ml−1 and suggests that the APP shifts from a community of PE and phycocyanin-rich picocyanobacteria and picoeukaryotes in winter to a PE-rich community in summer

Identification of factors constraining nitrate assimilation in Lake Superior, Laurentian Great Lakes

Despite a well-documented rise in nitrate concentration over the past century, Lake Superior has retained an oligotrophic character. In part, this status results from physical attributes of the lake including low temperatures and prolonged isothermy, resulting in deep-mixing and light limitation which constrain primary production. Lake Superior is also phosphorus deficient which limits phytoplankton growth. We conducted large (20 l) volume factorial bioassay experiments to assess the influence of light and nutrients (P, Fe) on nitrate assimilation by a Lake Superior chlorophyte alga. Bioassays seeded with the chlorophyte yielded a strong response to light resulting in the rapid depletion of nitrate. High light resulted in higher activities of the key N-assimilation enzyme nitrate reductase (NR) and increased algal biomass compared to low light treatments. NR activity was highly correlated with rates of nitrate incorporation in bioassays and field surveys suggesting that NR occupies a critical place in nitrate metabolism. In bioassays, the addition of nutrients (P, Fe) only slightly increased the rate at which nitrate became depleted. Parallel trials using a luminescent cyanobacterial bioreporter confirmed the lack of response by added nutrients supporting light as an important factor in constraining nitrate assimilation by phytoplankton in the lake. © 2013 Springer Science+Business Media Dordrecht