Project description:Chemical analysis of the compounds present in sediment, although informative, often is not indicative of the downstream biological effects that these contaminants exert on resident aquatic organisms. More direct molecular methods are needed to determine if marine life is affected by exposure to sediments. In this study, we used an aquatic multispecies microarray and q-PCR to investigate the effects on gene expression in juvenile sea bream (Sparus aurata) of two contaminated sediments defined as sediment 1 and 2 respectively, from marine areas in Northern Italy.
Project description:Crude oil is the one of the most important natural assets of humankind, yet it is a major environmental pollutant, in particular, in marine environments. One of the largest crude oil polluted areas in the word is the semi-enclosed Mediterranean Sea, where the metabolic potential of indigenous populations towards the chronic pollution at a large scale is yet to be defined, particularly in anaerobic and micro-anaerobic marine sites. Here, we provided a novel insight into the active microbial metabolism in sediments from three environments along the coastline of Italy. Microbial proteomes exhibited prevalence in anaerobic metabolism, not related to the biodegradation directly, suggesting the strong limitation by oxygen induced by the carbon overload. They also point at previously unrecognized metabolic coupling between methane and methanol utilizers as well as sulfur reducers in marine petroleum polluted sediments.
Project description:We have developed a 60-mer oligonucleotide multibacterial microarray for detection and expression profiling of biodegradative genes and bacterial diversity (16S rRNA gene) in different habitats contaminated with varieties of hazardous chemicals. The genes selected were involved in biodegradation and biotransformation of various groups of compounds viz. nitroaromatic compounds (148 genes), chloroaromatic compounds (75 genes), monoaromatic compounds (373 genes), polyaromatic hydrocarbons (174 genes), pesticides/ herbicides (34 genes), alkanes/aliphatics (185 genes) and heavy metals (68 genes), which covered a total number of 133 chemicals. The efficiency (specificity, detection sensitivity) of the developed array was evaluated using the labeled genomic DNA of pure bacterial strains, Escherichia coli DH5M-NM-1 and Sphingomonas sp. strain NM-05 (involved in the biodegradation of M-NM-3-hexachlorohexane isolated from IPL, Lucknow) at different concentrations of 300ng, 500ng, 800ng, 1000ng and 1250ng. The specificity of the developed array was further validated using mixed cultures containing three strains (Sphingomonas sp. strain NM-05, Rhodococcus sp. strain RHA1 and Bordetella sp. strain IITR-02) involved in biodegradation of M-NM-3-hexachlorohexane, biphenyl and chlorobenzenes respectively. The mixed culture also contained non-target/non-degrader strains (E. coli DHM-NM-1, E.coli BL21 and E.coli K12 NCTC50192). The developed array was applied for profiling using the total soil DNA in five contaminated habitats of north India, viz. chloroaromatic chemicals contaminated site (India Pesticide Limited, Chinhat, Lucknow), a river sediments (Gomti river sediment, Lucknow), heavy metal industry dump site (Jajmau industrial area Kanpur), a effluent treatment plant (CETP along Ganges river near Kanpur), and an oil refinery (Mathura oil refinery). Hybridization of 16S rRNA probes revealed the presence of bacteria similar to well characterized genera involved in biodegradation of pollutants. Genes involved in complete degradation pathways for hexachlorocyclohexane (lin), 1,2,4-trichlorobenzene (tcb), naphthalene (nah), phenol (mph), biphenyl (bph), benzene (ben), toluene (tbm), xylene (xyl), phthalate (pht), Salicylate (sal) and resistance to mercury (mer) were detected with highest intensity. The most abundant genes belonged to hydroxylases, monooxygenases and dehydrogenases which were present in all the five samples. Many compound specific genes which initiate the degradation pathway were also detected. Thus, the array developed and validated here may be useful in assessing the biodegradative potential and composition of environmentally useful bacteria in hazardous ecosystems. Agilent one-color CGH experiment,Organism: Genotypic designed Agilent-17159 Genotypic designed Agilent Multibacterial 8x15k Array , Labeling kit: Agilent Genomic DNA labeling Kit (Part Number: 5190-0453)
Project description:The marine bacterium Rhodococcus erythropolis PR4 was demonstrated to be able for assimilation/biodegradation of hydrocarbons. Not just the chromosome but two large plasmids provide versatile enzyme sets involved in many metabolic pathways. In order to identify the key elements involved in biodegradation of the model compound, hexadecane, and diesel oil, we performed whole transcriptome analysis on cells grown in the presence of n-hexadecane and diesel oil. Sodium acetate grown cells were used as control. The final goal of the project is a comparative transcriptomic analysis of Rhodococcus erythropolis PR4 cells grown on acetate, on the model compound: hexadecane and the real substrate: diesel oil.
Project description:We have developed a 60-mer oligonucleotide multibacterial microarray for detection and expression profiling of biodegradative genes and bacterial diversity (16S rRNA gene) in different habitats contaminated with varieties of hazardous chemicals. The genes selected were involved in biodegradation and biotransformation of various groups of compounds viz. nitroaromatic compounds (148 genes), chloroaromatic compounds (75 genes), monoaromatic compounds (373 genes), polyaromatic hydrocarbons (174 genes), pesticides/ herbicides (34 genes), alkanes/aliphatics (185 genes) and heavy metals (68 genes), which covered a total number of 133 chemicals. The efficiency (specificity, detection sensitivity) of the developed array was evaluated using the labeled genomic DNA of pure bacterial strains, Escherichia coli DH5α and Sphingomonas sp. strain NM-05 (involved in the biodegradation of γ-hexachlorohexane isolated from IPL, Lucknow) at different concentrations of 300ng, 500ng, 800ng, 1000ng and 1250ng. The specificity of the developed array was further validated using mixed cultures containing three strains (Sphingomonas sp. strain NM-05, Rhodococcus sp. strain RHA1 and Bordetella sp. strain IITR-02) involved in biodegradation of γ-hexachlorohexane, biphenyl and chlorobenzenes respectively. The mixed culture also contained non-target/non-degrader strains (E. coli DHα, E.coli BL21 and E.coli K12 NCTC50192). The developed array was applied for profiling using the total soil DNA in five contaminated habitats of north India, viz. chloroaromatic chemicals contaminated site (India Pesticide Limited, Chinhat, Lucknow), a river sediments (Gomti river sediment, Lucknow), heavy metal industry dump site (Jajmau industrial area Kanpur), a effluent treatment plant (CETP along Ganges river near Kanpur), and an oil refinery (Mathura oil refinery). Hybridization of 16S rRNA probes revealed the presence of bacteria similar to well characterized genera involved in biodegradation of pollutants. Genes involved in complete degradation pathways for hexachlorocyclohexane (lin), 1,2,4-trichlorobenzene (tcb), naphthalene (nah), phenol (mph), biphenyl (bph), benzene (ben), toluene (tbm), xylene (xyl), phthalate (pht), Salicylate (sal) and resistance to mercury (mer) were detected with highest intensity. The most abundant genes belonged to hydroxylases, monooxygenases and dehydrogenases which were present in all the five samples. Many compound specific genes which initiate the degradation pathway were also detected. Thus, the array developed and validated here may be useful in assessing the biodegradative potential and composition of environmentally useful bacteria in hazardous ecosystems.
Project description:The marine bacterium Rhodococcus erythropolis PR4 was demonstrated to be able for assimilation/biodegradation of hydrocarbons. Not just the chromosome but two large plasmids provide versatile enzyme sets involved in many metabolic pathways. In order to identify the key elements involved in biodegradation of the model compound, hexadecane, and diesel oil, we performed whole transcriptome analysis on cells grown in the presence of n-hexadecane and diesel oil. Sodium acetate grown cells were used as control. The final goal of the project is a comparative transcriptomic analysis of Rhodococcus erythropolis PR4 cells grown on acetate, on the model compound: hexadecane and the real substrate: diesel oil. Comparative transcriptomics of Rhodococcus erythropolis PR4 grown on n-hexadecane, diesel oil, and sodium acetate.
Project description:The application of chemical dispersants during marine oil spills can affect the community composition and activity of native marine microorganisms. Several studies have indicated that certain marine hydrocarbon-degrading bacteria, such as Marinobacter spp., can be inhibited by chemical dispersants, resulting in lower abundances and/or reduced hydrocarbon-biodegradation rates. In this respect, a major knowledge gap exists in understanding the mechanisms underlying these observed physiological effects. Here, we performed comparative proteomics of the Deepwater Horizon isolate Marinobacter sp. TT1 grown under different conditions that varied regarding the supplied carbon sources (pyruvate vs. n-hexadecane) and whether or not dispersant (Corexit EC9500A) was added, or that contained crude oil in the form of a water-accommodated fraction (WAF) or chemically-enhanced WAF (CEWAF). We characterized the proteins associated with alkane metabolism and alginate biosynthesis in strain TT1, report on its potential for aromatic hydrocarbon biodegradation and present a proposed metabolism of Corexit components as carbon substrates for the strain. Our findings implicate Corexit in affecting hydrocarbon metabolism, chemotactic motility, biofilm formation, and inducing solvent tolerance mechanisms like efflux pumps in strain TT1. This study provides novel insights into dispersant impacts on microbial hydrocarbon degraders that should be taken into consideration for future oil spill response actions.
Project description:The zebrafish embryo has repeatedly proved to be a useful model for the analysis of effects by environmental toxicants. This proof-of-concept study was performed to investigate if an approach combining mechanism-specific bioassays with microarray techniques can obtain more in-depth insights into the ecotoxicity of complex pollutant mixtures as present, e.g., in sediment extracts. For this end, altered gene expression was compared to data from established bioassays as well as to results from chemical analysis. Microarray analysis revealed several classes of significantly regulated genes which could to a considerably extend be related to the hazard potential. Results indicate that potential classes of contaminants can be assigned to sediment extracts by both classical biomarker genes and corresponding expression profile analyses of known substances. However, it is difficult to distinguish between specific responses and more universal detoxification of the organism. Microarray analysis were performed with early life stages of zebrafish exposed to 2 sediment extracts from the Upper part of the River Rhine, Germany. The expression profile as then compared to the expression pattern of model toxicants, such as, 4-chloroaniline, Cadmium, DDT, TCDD, and Valproic acid (Gene Expression Omnibus Series GSE9357). Additionally, combining mechanism-specific bioassays as well as chemical analysis of the sediments to the gene expression data has contributed to a more comprehensive view on the hazard potential of the sediments.
Project description:Toxicity of river sediments are assessed using whole sediment toxicity tests with benthic organisms. The challenge, however, is the differentiation between multiple effects caused by complex contaminant mixtures and the unspecific toxicity endpoints such as survival, growth or reproduction. Moreover, natural sediment properties, such as grain size distribution and organic carbon content, can influence the test parameters by masking pollutant toxicity. The use of gene expression profiling facilitates the identification of transcriptional changes at the molecular level that are specific to the bioavailable fraction of pollutants. The nematode Caenorhabditis elegans is ideally suited for these purposes, as (i) it can be exposed to whole sediments, and (ii) its genome is fully sequenced and widely annotated. In this pilot study we exposed C. elegans for 48 h to three sediments varying in degree of contamination with e.g. heavy metals and organic pollutants. Following the exposure period, gene expression was profiled using a whole genome DNA-microarray approach.