Dataset Information

Earthworm Lumbricus rubellus MT-2: Metal Binding and Protein Folding of a True Cadmium-MT.

ABSTRACT: Earthworms express, as most animals, metallothioneins (MTs)-small, cysteine-rich proteins that bind d(10) metal ions (Zn(II), Cd(II), or Cu(I)) in clusters. Three MT homologues are known for Lumbricus rubellus, the common red earthworm, one of which, wMT-2, is strongly induced by exposure of worms to cadmium. This study concerns composition, metal binding affinity and metal-dependent protein folding of wMT-2 expressed recombinantly and purified in the presence of Cd(II) and Zn(II). Crucially, whilst a single Cd?wMT-2 species was isolated from wMT-2-expressing E. coli cultures supplemented with Cd(II), expressions in the presence of Zn(II) yielded mixtures. The average affinities of wMT-2 determined for either Cd(II) or Zn(II) are both within normal ranges for MTs; hence, differential behaviour cannot be explained on the basis of overall affinity. Therefore, the protein folding properties of Cd- and Zn-wMT-2 were compared by ¹H NMR spectroscopy. This comparison revealed that the protein fold is better defined in the presence of cadmium than in the presence of zinc. These differences in folding and dynamics may be at the root of the differential behaviour of the cadmium- and zinc-bound protein in vitro, and may ultimately also help in distinguishing zinc and cadmium in the earthworm in vivo.

SUBMITTER: Kowald GR

PROVIDER: S-EPMC4730310 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Publications

Earthworm Lumbricus rubellus MT-2: Metal Binding and Protein Folding of a True Cadmium-MT.

Kowald Gregory R GR Stürzenbaum Stephen R SR Blindauer Claudia A CA

International journal of molecular sciences 20160105 1

Earthworms express, as most animals, metallothioneins (MTs)-small, cysteine-rich proteins that bind d(10) metal ions (Zn(II), Cd(II), or Cu(I)) in clusters. Three MT homologues are known for Lumbricus rubellus, the common red earthworm, one of which, wMT-2, is strongly induced by exposure of worms to cadmium. This study concerns composition, metal binding affinity and metal-dependent protein folding of wMT-2 expressed recombinantly and purified in the presence of Cd(II) and Zn(II). Crucially, wh ...[more]

PMID: 26742040

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Project description:BackgroundPopulations of the earthworm, Lumbricus rubellus, are commonly found across highly contaminated former mine sites and are considered to have under-gone selection for mitigating metal toxicity. Comparison of adapted populations with those found on less contaminated soils can provide insights into ecological processes that demonstrate the long-term effects of soil contamination. Contemporary sequencing methods allow for portrayal of demographic inferences and highlight genetic variation indicative of selection at specific genes. Furthermore, the occurrence of L. rubellus lineages across the UK allows for inferences of mechanisms associated with drivers of speciation and local adaptation.ResultsUsing RADseq, we were able to define population structure between the two lineages through the use of draft genomes for each, demonstrating an absence of admixture between lineages and that populations over extensive geographic distances form discrete populations. Between the two British lineages, we were able to provide evidence for selection near to genes associated with epigenetic and morphological functions, as well as near a gene encoding a pheromone. Earthworms inhabiting highly contaminated soils bare close genomic resemblance to those from proximal control soils. We were able to define a number of SNPs that largely segregate populations and are indicative of genes that are likely under selection for managing metal toxicity. This includes calcium and phosphate-handling mechanisms linked to lead and arsenic contaminants, respectively, while we also observed evidence for glutathione-related mechanisms, including metallothionein, across multiple populations. Population genomic end points demonstrate no consistent reduction in nucleotide diversity, or increase in inbreeding coefficient, relative to history of exposure.ConclusionsThough we can clearly define lineage membership using genomic markers, as well as population structure between geographic localities, it is difficult to resolve markers that segregate entirely between populations in response to soil metal concentrations. This may represent a highly variable series of traits in response to the heterogenous nature of the soil environment, but ultimately demonstrates the maintenance of lineage-specific genetic variation among local populations. L. rubellus appears to provide an exemplary system for exploring drivers for speciation, with a continuum of lineages coexisting across continental Europe, while distinct lineages exist in isolation throughout the UK.

Project description:BackgroundThe accurate delimitation of species is essential to numerous areas of biological research. An unbiased assessment of the diversity, including the cryptic diversity, is of particular importance for the below ground fauna, a major component of global biodiversity. On the British Isles, the epigeic earthworm Lumbricus rubellus, which is a sentinel species in soil ecotoxicology, consists of two cryptic taxa that are differentiated in both the nuclear and the mitochondrial (mtDNA) genomes. Recently, several deeply divergent mtDNA lineages were detected in mainland Europe, but whether these earthworms also constitute cryptic species remains unclear. This information is important from an evolutionary perspective, but it is also essential for the interpretation and the design of ecotoxicological projects. In this study, we used genome-wide RADseq data to assess the reproductive isolation of the divergent mitochondrial lineages of L. rubellus that occur in sympatry in multiple localities in Central Europe.ResultsWe identified five divergent (up to 16 % net p-distance) mitochondrial lineages of L. rubellus in sympatry. Because the clustering of the RADseq data was according to the population of origin and not the mtDNA lineage, reproductive isolation among the mtDNA lineages was not likely. Although each population contained multiple mtDNA lineages, subdivisions within the populations were not observed for the nuclear genome. The lack of fixed differences and sharing of the overwhelming majority of nuclear polymorphisms between localities, indicated that the populations did not constitute allopatric species. The nucleotide diversity within the populations was high, 0.7-0.8 %.ConclusionsThe deeply divergent mtDNA sympatric lineages of L. rubellus in Central Europe were not reproductively isolated groups. The earthworm L. rubellus, which is represented by several mtDNA lineages in continental Europe, apparently is a single highly polymorphic species rather than a complex of several cryptic species. This study demonstrated the critical importance of the use of multilocus nuclear data for the unbiased assessment of cryptic diversity and for the delimitation of species in soil invertebrates.

Project description:The microbial populations in no-till agricultural soil and casts of the earthworm Lumbricus rubellus were examined by culturing and molecular methods. Clone libraries of the 16S rRNA genes were prepared from DNA isolated directly from the soil and earthworm casts. Although no single phylum dominated the soil library of 95 clones, the largest numbers of clones were from Acidobacteria (14%), Cytophagales (13%), Chloroflexi (8%), and gamma-Proteobacteria (8%). While the cast clone library of 102 clones was similar to the soil library, the abundances of several taxa were different. Representatives of the Pseudomonas genus as well as the Actinobacteria and Firmicutes increased in number, and one group of unclassified organisms found in the soil library was absent in the cast library. Likewise, soil and cast archaeal 16S rRNA gene libraries were similar, although the abundances of some groups were different. Two hundred and thirty aerobic bacteria were also isolated on general heterotrophic media from casts, burrows, and soil. The cast isolates were both phenotypically and genotypically different from the soil isolates. The cast isolates were more likely to reduce nitrate, grow on acetate and Casamino Acids, and utilize fewer sugars than the soil isolates. On the basis of their ribotypes, the cast isolates were dominated by Aeromonas spp. (28%), which were not found in the soil isolates, and other gamma-Proteobacteria (49%). In contrast, the soil isolates were mostly Actinobacteria (53%), Firmicutes (16%), and gamma-Proteobacteria (19%). Isolates obtained from the sides of earthworm burrows were not different from the soil isolates. Diversity indices for the collections of isolates as well as rRNA gene libraries indicated that the species richness and evenness were decreased in the casts from their levels in the soil. These results were consistent with a model where a large portion of the microbial population in soil passes through the gastrointestinal tract of the earthworm unchanged while representatives of some phyla increase in abundance.

Dataset Information

Earthworm Lumbricus rubellus MT-2: Metal Binding and Protein Folding of a True Cadmium-MT.

Publications

Earthworm Lumbricus rubellus MT-2: Metal Binding and Protein Folding of a True Cadmium-MT.

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