Sheela Reuben

MetDAT: a modular and workflow-based free online pipeline for mass spectrometry data processing, analysis and interpretation

SUMMARY Analysis of high throughput metabolomics experiments is a resource-intensive process that includes pre-processing, pre-treatment and post-processing at each level of experimental hierarchy. We developed an interactive user-friendly online software called Metabolite Data Analysis Tool (MetDAT) for mass spectrometry data. It offers a pipeline of tools for file handling, data pre-processing, univariate and multivariate statistical analyses, database searching and pathway mapping. Outputs are produced in the form of text and high-quality images in real-time. MetDAT allows users to combine data management and experiment-centric workflows for optimization of metabolomics methods and metabolite analysis. AVAILABILITY MetDAT is available free for academic use from http://smbl.nus.edu.sg/METDAT2/. CONTACT sanjay@nus.edu.sg

The Promoter Signatures in Rice LEA Genes Can Be Used to Build a Co-expressing LEA Gene Network

Coordinated transcriptional modulation of large gene sets depends on the combinatorial use of cis-regulatory motifs in promoters. We postulate that promoter content similarities are diagnostic for co-expressing genes that function coherently during specific cellular responses. To find the co-expressing genes we propose an ab initio method that identifies motif families in promoters of target gene groups, map these families to the promoters of all genes in the genome, and determine the best matches of each of the target group gene promoters with all other promoters. When the method was tested in rice starting from a group of co-expressing Late Embryogenesis Abundant (LEA) genes, we obtained a promoter similarity-based network that contained candidate genes that could plausibly complement the function of LEA genes. Importantly, 73.36% of 244 genes predicted by our method were experimentally confirmed to co-express with the LEA genes in maturing rice embryos, making this methodology a promising tool for biological systems analyses.

Ecogenomics reveals metals and land-use pressures on microbial communities in the waterways of a megacity.

Networks of engineered waterways are critical in meeting the growing water demands in megacities. To capture and treat rainwater in an energy-efficient manner, approaches can be developed for such networks that use ecological services from microbial communities. Traditionally, engineered waterways were regarded as homogeneous systems with little responsiveness of ecological communities and ensuing processes. This study provides ecogenomics-derived key information to explain the complexity of urban aquatic ecosystems in well-managed watersheds with densely interspersed land-use patterns. Overall, sedimentary microbial communities had higher richness and evenness compared to the suspended communities in water phase. On the basis of PERMANOVA analysis, variation in structure and functions of microbial communities over space within same land-use type was not significant. In contrast, this difference was significant between different land-use types, which had similar chemical profiles. Of the 36 environmental parameters from spatial analysis, only three metals, namely potassium, copper and aluminum significantly explained between 7% and 11% of the variation in taxa and functions, based on distance-based linear models (DistLM). The ecogenomics approach adopted here allows the identification of key drivers of microbial communities and their functions at watershed-scale. These findings can be used to enhance microbial services, which are critical to develop ecologically friendly waterways in rapidly urbanizing environments.

Combination of synchrotron radiation-based Fourier transforms infrared microspectroscopy and confocal laser scanning microscopy to understand spatial heterogeneity in aquatic multispecies biofilms.

Understanding the spatial heterogeneity within environmental biofilms can provide an insight into compartmentalization of different functions in biofilm communities. We used a non-destructive and label-free method by combining Synchrotron Radiation-based Fourier Transform Infrared Microspectroscopy (SR-FTIR) with Confocal Laser Scanning Microscopy (CLSM) to distinguish the spatial chemical changes within multispecies biofilms grown from natural storm waters in flow cells. Among the different surfaces tested for biofilm growth and optimal imaging, mylar membranes were most suited and it enabled successful spatial infrared imaging of natural biofilms for obtaining reliable and interpretable FTIR spectra. Time series analysis of biofilm growth showed that influx of water during biofilm growth, results in significant changes in biofilm formation. Early biofilms showed active nutrient acquisition and desiccation tolerance mechanisms corresponding with accumulation of secreted proteins. Statistical approach used for the evaluation of chemical spectra allowed for clustering and classification of various regions of the biofilm. Microheterogeneity was observed in the polymeric components of the biofilm matrix, including cellulose, glycocalyx and dextran-like molecules. Fructan and glycan-rich regions were distinguishable and glycocalyx was abundant in the strongly adhering peripheral regions of biofilms. Inner core showed coexistence of oxygen dimers and ferrihydrite that will likely support growth of Fe (II)-oxidising bacteria. The combined SR-FTIR microspectroscopy and CSLM approach for complex natural biofilms described here will be useful both in understanding heterogeneity of matrix components and in correlating functions of juxtaposed microbial species in complex natural biofilms with physicochemical microenvironment to which they are exposed.

A bacterial quercetin oxidoreductase QuoA-mediated perturbation in the phenylpropanoid metabolic network increases lignification with a concomitant decrease in phenolamides in Arabidopsis

Metabolic perturbations by a gain-of-function approach provide a means to alter steady states of metabolites and query network properties, while keeping enzyme complexes intact. A combination of genetic and targeted metabolomics approach was used to understand the network properties of phenylpropanoid secondary metabolism pathways. A novel quercetin oxidoreductase, QuoA, from Pseudomonas putida, which converts quercetin to naringenin, thus effectively reversing the biosynthesis of quercetin through a de novo pathway, was expressed in Arabidopsis thaliana. QuoA transgenic lines selected for low, medium, and high expression levels of QuoA RNA had corresponding levels of QuoA activity and hypocotyl coloration resulting from increased anthocyanin accumulation. Stems of all three QuoA lines had increased tensile strength resulting from increased lignification. Sixteen metabolic intermediates from anthocyanin, lignin, and shikimate pathways had increased accumulation, of which 11 paralleled QuoA expression levels in the transgenic lines. The concomitant upregulation of the above pathways was explained by a significant downregulation of the phenolamide pathway and its precursor, spermidine. In a tt6 mutant line, lignifications as well as levels of the lignin pathway metabolites were much lower than those of QuoA transgenic lines. Unlike QuoA lines, phenolamides and spermidine were not affected in the tt6 line. Taken together, these results suggest that phenolamide pathway plays a major role in directing metabolic intermediates into the lignin pathway. Metabolic perturbations were accompanied by downregulation of five genes associated with branch-point enzymes and upregulation of their corresponding products. These results suggest that gene–metabolite pairs are likely to be co-ordinately regulated at critical branch points. Thus, these perturbations by a gain-of-function approach have uncovered novel properties of the phenylpropanoid metabolic network.

Bacterial diversity on different surfaces in urban freshwater

Microbial loads in freshwater systems have important implications in biogeochemical cycling in urban environments. Immersed surfaces in freshwaters provide surfaces for bacterial attachment and growth. Microorganisms that adhere initially to these surfaces play a critical role in biofilm formation and sustenance. Currently, there is little understanding on the type of organisms that initially adhere to different surfaces in urban canals. In this study, water from an urban stormwater canal was employed to allow bacteria to attach to different surfaces in a flowcell apparatus and understand the differences and changes in bacterial community structure. Bacterial communities were highly diverse on different surfaces as indicated by Jaccards indices of 0.14-0.56. Bacteria on aluminium were the most diverse and on Plexiglas the least. Bacterial communities were highly dynamic in the early attachment phase and it changed by 59% between 3 and 6 h on aluminium. Specificity of attachment to surfaces was observed for some bacteria. Judicious use of materials in urban aquatic environment would help mitigate microbial load in urban waters.

datPAV—an online processing, analysis and visualization tool for exploratory investigation of experimental data

SUMMARY Data processing, analysis and visualization (datPAV) is an exploratory tool that allows experimentalist to quickly assess the general characteristics of the data. This platform-independent software is designed as a generic tool to process and visualize data matrices. This tool explores organization of the data, detect errors and support basic statistical analyses. Processed data can be reused whereby different step-by-step data processing/analysis workflows can be created to carry out detailed investigation. The visualization option provides publication-ready graphics. Applications of this tool are demonstrated at the web site for three cases of metabolomics, environmental and hydrodynamic data analysis. AVAILABILITY datPAV is available free for academic use at http://www.sdwa.nus.edu.sg/datPAV/.

Reduction of nutrient contaminants into shallow eutrophic waters through vegetated treatment beds

One of the most effective mitigative approaches to eutrophication is the reduction of nutrient loading into water bodies. Bioremediation presents an economically viable and ecologically sustainable technology to nutrient pollution control taking advantage of the remarkable ability of plants and their associated microbial community to assimilate and remove nutrients from the environment. In this study, four emergent macrophytes (Cyperus haspan, Pandanus amaryllifolius, Pontederia cordata and Thalia geniculata) and two floating plants (Hygroryza aristata and Pistia stratiotes) were deployed in bank-side treatment beds and comparatively assessed for their remediative capabilities for nutrient control. P. stratiotes exhibited the highest removal efficiency for both nitrate and phosphate among the six plant species studied. Emergent macrophytes, P. amaryllifolius, C. haspan and P. cordata, were also found to be highly effective in nutrient uptake exhibiting removal efficiencies up to 100%. With the exception of T. geniculata, depletion of nutrients as a result of plant uptake significantly impeded the natural colonization of algae invariably leading to improvements in water quality in terms of turbidity and pH. Suppression of algae proliferation by T. geniculata was not preceded by a reduction in nutrient concentrations suggesting that T. geniculata may be directly inhibiting algal growth through allelopathy.

RhizoFlowCell system reveals early effects of micropollutants on aquatic plant rhizosphere

In aquatic systems, one of the non-destructive ways to quantify toxicity of contaminants to plants is to monitor changes in root exudation patterns. In aquatic conditions, monitoring and quantifying such changes are currently challenging because of dilution of root exudates in water phase and lack of suitable instrumentation to measure them. Exposure to pollutants would not only change the plant exudation, but also affect the microbial communities that surround the root zone, thereby changing the metabolic profiles of the rhizosphere. This study aims at developing a device, the RhizoFlowCell, which can quantify metabolic response of plants, as well as changes in the microbial communities, to give an estimate of the stress to which the rhizosphere is exposed. The usefulness of RhizoFlowCell is demonstrated using naphthalene as a test pollutant. Results show that RhizoFlowCell system is useful in quantifying the dynamic metabolic response of aquatic rhizosphere to determine ecosystem health.

In situ removal of dissolved and suspended contaminants from a eutrophic pond using hybrid sand-filter

In this study, in situ hybrid sand filters were designed to remove dissolved and suspended contaminants from eutrophic pond. Currently, there are no attempts made to eradicate dissolved as well as suspended contaminants from eutrophic water system in a single step. Monitoring studies revealed that examined pond contain high chlorophyll-a content (101.8 μg L−1), turbidity (39.5 NTU) and total dissolved solids concentration (0.04 g L−1). Samples were further exposed to extensive water quality analysis, which include examining physicochemical parameters (pH, conductivity, total dissolved solids, salinity, turbidity and chlorophyll-a), metals (Na, K, Ca, Mg, Al, Fe, Cu, Cd, Pb, Zn, Cr, and Ni) and anions (NO3, NO2, PO4, SO4, Cl, F and Br). To tackle pollutants, filtration system was designed to comprise of several components including fine sand, coarse sand/sorbent mix and gravel from top to bottom loaded in fiberglass tanks. All the filters (activated carbon, Sargassum and zeolite) completely removed algal biomass and showed potential to decrease pH during entire operational period of 20 h at 120 L h−1. To examine the efficiency of filters in adverse conditions, the pond water was spiked with heavy metals (Cu, Cd, Pb, Zn, Cr, and Ni). Of the different filter systems, Sargassum-loaded filter performed exceedingly well with concentrations of heavy metals never exceeded the Environmental protection agency regulations for freshwater limits during total operational period. The total uptake capacities at the end of the fifth event were 24.9, 20.5, 0.58, 5.2, 0.091 and 2.8 mg/kg for Cr, Ni, Cu, Zn, Cd and Pb, respectively.