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:While more commonly associated with plants than microbes, diterpenoid natural products have been reported to have profound effects in marine microbe-microbe interactions. Intriguingly, the genome of the marine bacterium Salinispora arenicola CNS-205 contains a putative diterpenoid biosynthetic operon, terp1. Here recombinant expression studies are reported, indicating that this three-gene operon leads to the production of isopimara-8,15-dien-19-ol (4). Although 4 is not observed in pure cultures of S. arenicola, it is plausible that the terp1 operon is only expressed under certain physiologically relevant conditions such as in the presence of other marine organisms.
Project description:Three new cyclohexadepsipeptides, arenamides A-C (1-3), were isolated from the fermentation broth of a marine bacterial strain identified as Salinispora arenicola. The planar structures of these compounds were assigned by detailed interpretation of NMR and MS/MS spectroscopic data. The absolute configurations of the amino acids, and those of the chiral centers on the side chain, were established by application of the Marfey and modified Mosher methods. The effect of arenamides A and B on NFkappaB activity was studied with stably transfected 293/NFkappaB-Luc human embryonic kidney cells induced by treatment with tumor necrosis factor (TNF). Arenamides A (1) and B (2) blocked TNF-induced activation in a dose- and time-dependent manner with IC(50) values of 3.7 and 1.7 microM, respectively. In addition, the compounds inhibited nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production with lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Moderate cytotoxicity was observed with the human colon carcinoma cell line HCT-116, but no cytotoxic effect was noted with cultured RAW cells. Taken together, these data suggest that the chemoprevention and anti-inflammatory characteristics of arenamides A and B warrant further investigation.
Project description:The molecular fingerprinting technique terminal-restriction fragment length polymorphism (T-RFLP) was used in combination with sequence-based approaches to evaluate the geographic distribution of secondary metabolite biosynthetic genes in strains of the marine actinomycete Salinispora arenicola. This study targeted ketosynthase (KS) domains from type I polyketide synthase (PKS) genes and revealed four distinct clusters, the largest of which was comprised of strains from all six global locations sampled. The remaining strains fell into three smaller clusters comprised of strains derived entirely from the Red Sea, the Sea of Cortez, or around the Island of Guam. These results reveal variation in the secondary metabolite gene collectives maintained by strains that are largely clonal at the 16S rRNA level. The location specificities of the three smaller clusters provide evidence that collections of secondary metabolite genes in subpopulations of S. arenicola are endemic to these locations. Cloned KS sequences support the maintenance of distinct sets of biosynthetic genes in the strains associated with each cluster and include four that had not previously been detected in S. arenicola. Two of these new sequences were observed only in strains derived from Guam or the Sea of Cortez. Transcriptional analysis of one of the new KS sequences in conjunction with the production of the polyketide arenicolide A supports a link between this sequence and the associated biosynthetic pathway. From the perspective of natural product discovery, these results suggest that screening populations from distant locations can enhance the discovery of new natural products and provides further support for the use of molecular fingerprinting techniques, such as T-RFLP, to rapidly identify strains that possess distinct sets of biosynthetic genes.
Project description:Chemical evaluation of the saline fermentation broth of several strains of the obligate marine actinomycete Salinispora arenicola has led to the identification of three new macrolide polyketides designated arenicolides A-C (1-3). The planar structures, elucidated via spectroscopic and chemical methods, consist of 26-membered polyunsaturated macrolactones containing repeating vicinal hydroxyl methoxyl moieties. The relative and absolute stereochemistries of 1-3 were assigned by a combination of J-based configurational analyses and chemical derivatization.
Project description:A variety of contaminants find their way to the marine sediments from different sources, and these contaminants can pose serious risks to the natural marine flora and fauna. For example, pyrethroids, which are a potent pesticide family, are often used in agriculture fields worldwide, and these find their way into the marine environment through run off. Further, pyrethroids are used in farmed Atlantic salmon cages in Chile, Great Britain and Norway. Ammonia is another contaminant that is used in agriculture in form of ammonia-rich fertilizer and can be carried during run-offs to localized rivers and streams. Ammonia is also detectable after emission of effluents from sewage treatment plants and industrial plants like oil refineries and meat processing plants. Contaminants may have short and long term effects on non-target organisms living in the water column or in the marine sediment. Importantly, the sediment ecosystem houses a variety of plants, animals and crustaceans, including the American lobster Homarus americanus. Lobster is the most fished crustacean in New Brunswick and Quebec and its resale and exportation produced over $1.6 billion in 2011. Due to its economic and environmental importance, it is essential to study the effects of contaminants present in its ecosystem. Sediment samples are often used as pollution markers during toxicity testing due to their tendency to accumulate hydrophobic contaminants. To better understand the possible effects of contaminants in sediment, a total gene expression study was developed using the marine amphipod Eohaustorius estuarius. A 10 day spike-in exposure was performed using ammonia and two pyrethroids, namely cypermethrin and deltamethrin. As pyrethroids and ammonia are known to have vastly different mechanisms of action in living organisms, we compared global gene expression patterns following exposure to ammonia against the patterns observed following exposure to pyrethroids. Total gene expression was measured by oligonucleotide microarray. The expression of five genes of interest involved in different biological processes such as metabolism, transcription, translation, immunity and stress, which were found to be differently expressed by microarray, was validated by RT-qPCR. A set of genes was identified that showed differential expression levels in a treatment-dependent manner, thus further highlighting the different mechanisms of action of ammonia and pyrethroids in the marine sediment. This study provides a proof of concept for the use of DNA microarrays with model crustaceans for the study of marine sediment contaminants.
Project description:A variety of contaminants find their way to the marine sediments from different sources, and these contaminants can pose serious risks to the natural marine flora and fauna. For example, pyrethroids, which are a potent pesticide family, are often used in agriculture fields worldwide, and these find their way into the marine environment through run off. Further, pyrethroids are used in farmed Atlantic salmon cages in Chile, Great Britain and Norway. Ammonia is another contaminant that is used in agriculture in form of ammonia-rich fertilizer and can be carried during run-offs to localized rivers and streams. Ammonia is also detectable after emission of effluents from sewage treatment plants and industrial plants like oil refineries and meat processing plants. Contaminants may have short and long term effects on non-target organisms living in the water column or in the marine sediment. Importantly, the sediment ecosystem houses a variety of plants, animals and crustaceans, including the American lobster Homarus americanus. Lobster is the most fished crustacean in New Brunswick and Quebec and its resale and exportation produced over $1.6 billion in 2011. Due to its economic and environmental importance, it is essential to study the effects of contaminants present in its ecosystem. Sediment samples are often used as pollution markers during toxicity testing due to their tendency to accumulate hydrophobic contaminants. To better understand the possible effects of contaminants in sediment, a total gene expression study was developed using the marine amphipod Eohaustorius estuarius. A 10 day spike-in exposure was performed using ammonia and two pyrethroids, namely cypermethrin and deltamethrin. As pyrethroids and ammonia are known to have vastly different mechanisms of action in living organisms, we compared global gene expression patterns following exposure to ammonia against the patterns observed following exposure to pyrethroids. Total gene expression was measured by oligonucleotide microarray. The expression of five genes of interest involved in different biological processes such as metabolism, transcription, translation, immunity and stress, which were found to be differently expressed by microarray, was validated by RT-qPCR. A set of genes was identified that showed differential expression levels in a treatment-dependent manner, thus further highlighting the different mechanisms of action of ammonia and pyrethroids in the marine sediment. This study provides a proof of concept for the use of DNA microarrays with model crustaceans for the study of marine sediment contaminants. This specific study is aimed at evaluating the effect of ammonia and pyrethroid exposure on E.estuarius and to identify possible biomarkers of these exposures.
Project description:Kentrophoros ciliates are found worldwide in coastal marine sediment, and have an obligate symbiosis with sulfur-oxidizing Gammaproteobacteria called Candidatus Kentron. The genomes of different Kentron species suggest that they are chemolithoheterotrophs, producing new biomass from organic carbon substrates. We investigated the metaproteome of Kentrophoros sp. H and its symbiont (from two sites in the Mediterranean), to verify that hypothesized pathways of carbon and energy metabolism are indeed expressed, and to estimate the stable isotope fractionation in biomass, which can be a signature of the carbon sources used by the organism.