Natural Chlordecone Degradation Revealed by Numerous Transformation Products Characterized in Key French West Indies Environmental Compartments.

Production and use of the insecticide chlordecone has caused long-term environmental pollution in the James River area and the French West Indies (FWI) that has resulted in acute human-health problems and a social crisis. High levels of chlordecone in FWI soils, even after its ban decades ago, and the absence of detection of transformation products (TPs), have suggested that chlordecone is virtually nonbiodegradable in the environment. Here, we investigated laboratory biodegradation, consisting of bacterial liquid cultures and microcosms inoculated with FWI soils, using a dual nontargeted GC-MS and LC-HRMS approach. In addition to previously reported, partly characterized hydrochlordecones and polychloroindenes (families A and B), we discovered 14 new chlordecone TPs, assigned to four families (B, C, D, and E). Organic synthesis and NMR analyses allowed us to achieve the complete structural elucidation of 19 TPs. Members of TP families A, B, C, and E were detected in soil, sediment, and water samples from Martinique and include 17 TPs not initially found in commercial chlordecone formulations. 2,4,5,6,7-Pentachloroindene was the most prominent TP, with levels similar to those of chlordecone. Overall, our results clearly show that chlordecone pollution extends beyond the parent chlordecone molecule and includes a considerable number of previously undetected TPs. Structural diversity of the identified TPs illustrates the complexity of chlordecone degradation in the environment and raises the possibility of extensive worldwide pollution of soil and aquatic ecosystems by chlordecone TPs.

Deciphering the evolution and metabolism of an anammox bacterium from a community genome.

Anaerobic ammonium oxidation (anammox) has become a main focus in oceanography and wastewater treatment. It is also the nitrogen cycle's major remaining biochemical enigma. Among its features, the occurrence of hydrazine as a free intermediate of catabolism, the biosynthesis of ladderane lipids and the role of cytoplasm differentiation are unique in biology. Here we use environmental genomics--the reconstruction of genomic data directly from the environment--to assemble the genome of the uncultured anammox bacterium Kuenenia stuttgartiensis from a complex bioreactor community. The genome data illuminate the evolutionary history of the Planctomycetes and allow us to expose the genetic blueprint of the organism's special properties. Most significantly, we identified candidate genes responsible for ladderane biosynthesis and biological hydrazine metabolism, and discovered unexpected metabolic versatility.

Clostridium sticklandii, a specialist in amino acid degradation:revisiting its metabolism through its genome sequence.

BACKGROUND: Clostridium sticklandii belongs to a cluster of non-pathogenic proteolytic clostridia which utilize amino acids as carbon and energy sources. Isolated by T.C. Stadtman in 1954, it has been generally regarded as a "gold mine" for novel biochemical reactions and is used as a model organism for studying metabolic aspects such as the Stickland reaction, coenzyme-B12- and selenium-dependent reactions of amino acids. With the goal of revisiting its carbon, nitrogen, and energy metabolism, and comparing studies with other clostridia, its genome has been sequenced and analyzed. RESULTS: C. sticklandii is one of the best biochemically studied proteolytic clostridial species. Useful additional information has been obtained from the sequencing and annotation of its genome, which is presented in this paper. Besides, experimental procedures reveal that C. sticklandii degrades amino acids in a preferential and sequential way. The organism prefers threonine, arginine, serine, cysteine, proline, and glycine, whereas glutamate, aspartate and alanine are excreted. Energy conservation is primarily obtained by substrate-level phosphorylation in fermentative pathways. The reactions catalyzed by different ferredoxin oxidoreductases and the exergonic NADH-dependent reduction of crotonyl-CoA point to a possible chemiosmotic energy conservation via the Rnf complex. C. sticklandii possesses both the F-type and V-type ATPases. The discovery of an as yet unrecognized selenoprotein in the D-proline reductase operon suggests a more detailed mechanism for NADH-dependent D-proline reduction. A rather unusual metabolic feature is the presence of genes for all the enzymes involved in two different CO2-fixation pathways: C. sticklandii harbours both the glycine synthase/glycine reductase and the Wood-Ljungdahl pathways. This unusual pathway combination has retrospectively been observed in only four other sequenced microorganisms. CONCLUSIONS: Analysis of the C. sticklandii genome and additional experimental procedures have improved our understanding of anaerobic amino acid degradation. Several specific metabolic features have been detected, some of which are very unusual for anaerobic fermenting bacteria. Comparative genomics has provided the opportunity to study the lifestyle of pathogenic and non-pathogenic clostridial species as well as to elucidate the difference in metabolic features between clostridia and other anaerobes.

Transformation of the recalcitrant pesticide chlordecone by Desulfovibrio sp.86 with a switch from ring-opening dechlorination to reductive sulfidation activity.

The insecticide chlordecone has been used in the French West Indies for decades, resulting in long term pollution, human health problems and social crisis. In addition to bacterial consortia and Citrobacter sp.86 previously described to transform chlordecone into three families of transformation products (A: hydrochlordecones, B: polychloroindenes and C: polychloroindenecarboxylic acids), another bacterium Desulfovibrio sp.86, showing the same abilities has been isolated and its genome was sequenced. Ring-opening dechlorination, leading to A, B and C families, was observed as previously described. Changing operating conditions in the presence of chlordecone gave rise to the formation of an unknown sulfur-containing transformation product instead of the aforementioned ones. Its structural elucidation enabled to conclude to a thiol derivative, which corresponds to an undocumented bacterial reductive sulfidation. Microbial experiments pointed out that the chlordecone thiol derivative was observed in anaerobiosis, and required the presence of an electron acceptor containing sulfur or hydrogen sulfide, in a confined atmosphere. It seems that this new reaction is also active on hydrochlordecones, as the 10-monohydrochlordecone A1 was transformed the same way. Moreover, the chlordecone thiol derivative called F1 was detected in several chlordecone contaminated mangrove bed sediments from Martinique Island, highlighting the environmental relevance of these results.

Genetic Analysis of Citrobacter sp.86 Reveals Involvement of Corrinoids in Chlordecone and Lindane Biotransformations.

Chlordecone (Kepone®) and γ-hexachlorocyclohexane (γ-HCH or lindane) have been used for decades in the French West Indies (FWI) resulting in long-term soil and water pollution. In a previous work, we have identified a new Citrobacter species (sp.86) that is able to transform chlordecone into numerous products under anaerobic conditions. No homologs to known reductive dehalogenases or other candidate genes were found in the genome sequence of Citrobacter sp.86. However, a complete anaerobic pathway for cobalamin biosynthesis was identified. In this study, we investigated whether cobalamin or intermediates of cobalamin biosynthesis was required for chlordecone microbiological transformation. For this purpose, we constructed a set of four Citrobacter sp.86 mutant strains defective in several genes belonging to the anaerobic cobalamin biosynthesis pathway. We monitored chlordecone and its transformation products (TPs) during long-term incubation in liquid cultures under anaerobic conditions. Chlordecone TPs were detected in the case of cobalamin-producing Citrobacter sp.86 wild-type strain but also in the case of mutants able to produce corrinoids devoid of lower ligand. In contrast, mutants unable to insert the cobalt atom in precorrin-2 did not induce any transformation of chlordecone. In addition, it was found that lindane, previously shown to be anaerobically transformed by Citrobacter freundii without evidence of a mechanism, was also degraded in the presence of the wild-type strain of Citrobacter sp.86. The lindane degradation abilities of the various Citrobacter sp.86 mutant strains paralleled chlordecone transformation. The present study shows the involvement of cobalt-containing corrinoids in the microbial degradation of chlorinated compounds with different chemical structures. Their increased production in contaminated environments could accelerate the decontamination processes.

A conserved gene cluster rules anaerobic oxidative degradation of L-ornithine.

For the ornithine fermentation pathway, described more than 70 years ago, genetic and biochemical information are still incomplete. We present here the experimental identification of the last four missing genes of this metabolic pathway. They encode L-ornithine racemase, (2R,4S)-2,4-diaminopentanoate dehydrogenase, and the two subunits of 2-amino-4-ketopentanoate thiolase. While described only for the Clostridiaceae to date, this pathway is shown to be more widespread.

Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium.

Acinetobacter sp. strain ADP1 is a nutritionally versatile soil bacterium closely related to representatives of the well-characterized Pseudomonas aeruginosa and Pseudomonas putida. Unlike these bacteria, the Acinetobacter ADP1 is highly competent for natural transformation which affords extraordinary convenience for genetic manipulation. The circular chromosome of the Acinetobacter ADP1, presented here, encodes 3325 predicted coding sequences, of which 60% have been classified based on sequence similarity to other documented proteins. The close evolutionary proximity of Acinetobacter and Pseudomonas species, as judged by the sequences of their 16S RNA genes and by the highest level of bidirectional best hits, contrasts with the extensive divergence in the GC content of their DNA (40 versus 62%). The chromosomes also differ significantly in size, with the Acinetobacter ADP1 chromosome <60% of the length of the Pseudomonas counterparts. Genome analysis of the Acinetobacter ADP1 revealed genes for metabolic pathways involved in utilization of a large variety of compounds. Almost all of these genes, with orthologs that are scattered in other species, are located in five major 'islands of catabolic diversity', now an apparent 'archipelago of catabolic diversity', within one-quarter of the overall genome. Acinetobacter ADP1 displays many features of other aerobic soil bacteria with metabolism oriented toward the degradation of organic compounds found in their natural habitat. A distinguishing feature of this genome is the absence of a gene corresponding to pyruvate kinase, the enzyme that generally catalyzes the terminal step in conversion of carbohydrates to pyruvate for respiration by the citric acid cycle. This finding supports the view that the cycle itself is centrally geared to the catabolic capabilities of this exceptionally versatile organism.

Microbial Degradation of a Recalcitrant Pesticide: Chlordecone.

Chlordecone (Kepone®) is a synthetic organochlorine insecticide (C10Cl10O) used worldwide mostly during the 1970 and 1980s. Its intensive application in the French West Indies to control the banana black weevil Cosmopolites sordidus led to a massive environmental pollution. Persistence of chlordecone in soils and water for numerous decades even centuries causes global public health and socio-economic concerns. In order to investigate the biodegradability of chlordecone, microbial enrichment cultures from soils contaminated by chlordecone or other organochlorines and from sludge of a wastewater treatment plant have been conducted. Different experimental procedures including original microcosms were carried out anaerobically over long periods of time. GC-MS monitoring resulted in the detection of chlorinated derivatives in several cultures, consistent with chlordecone biotransformation. More interestingly, disappearance of chlordecone (50 µg/mL) in two bacterial consortia was concomitant with the accumulation of a major metabolite of formula C9Cl5H3 (named B1) as well as two minor metabolites C10Cl9HO (named A1) and C9Cl4H4 (named B3). Finally, we report the isolation and the complete genomic sequences of two new Citrobacter isolates, closely related to Citrobacter amalonaticus, and that were capable of reproducing chlordecone transformation. Further characterization of these Citrobacter strains should yield deeper insights into the mechanisms involved in this transformation process.

Metabolic diversity among main microorganisms inside an arsenic-rich ecosystem revealed by meta- and proteo-genomics.

By their metabolic activities, microorganisms have a crucial role in the biogeochemical cycles of elements. The complete understanding of these processes requires, however, the deciphering of both the structure and the function, including synecologic interactions, of microbial communities. Using a metagenomic approach, we demonstrated here that an acid mine drainage highly contaminated with arsenic is dominated by seven bacterial strains whose genomes were reconstructed. Five of them represent yet uncultivated bacteria and include two strains belonging to a novel bacterial phylum present in some similar ecosystems, and which was named 'Candidatus Fodinabacter communificans.' Metaproteomic data unravelled several microbial capabilities expressed in situ, such as iron, sulfur and arsenic oxidation that are key mechanisms in biomineralization, or organic nutrient, amino acid and vitamin metabolism involved in synthrophic associations. A statistical analysis of genomic and proteomic data and reverse transcriptase-PCR experiments allowed us to build an integrated model of the metabolic interactions that may be of prime importance in the natural attenuation of such anthropized ecosystems.

A mutation of spastin is responsible for swellings and impairment of transport in a region of axon characterized by changes in microtubule composition.

Mutations of the spastin gene (Sp) are responsible for the most frequent autosomal dominant form of spastic paraplegia, a disease characterized by the degeneration of corticospinal tracts. We show that a deletion in the mouse Sp gene, generating a premature stop codon, is responsible for progressive axonal degeneration, restricted to the central nervous system, leading to a late and mild motor defect. The degenerative process is characterized by focal axonal swellings, associated with abnormal accumulation of organelles and cytoskeletal components. In culture, mutant cortical neurons showed normal viability and neurite density. However, they develop neurite swellings associated with focal impairment of retrograde transport. These defects occur near the growth cone, in a region characterized by the transition between stable microtubules rich in detyrosinated alpha-tubulin and dynamic microtubules composed almost exclusively of tyrosinated alpha-tubulin. Here, we show that the Sp mutation has a major impact on neurite maintenance and transport both in vivo and in vitro. These results highlight the link between spastin and microtubule dynamics in axons, but not in other neuronal compartments. In addition, it is the first description of a human neurodegenerative disease which involves this specialized region of the axon.

Mutations of SPG4 are responsible for a loss of function of spastin, an abundant neuronal protein localized in the nucleus.

Mutations of spastin are responsible for the most common autosomal dominant form of hereditary spastic paraplegia (AD-HSP), a disease characterized by axonal degeneration of corticospinal tracts and posterior columns. Generation of polyclonal antibodies specific to spastin has revealed two isoforms of 75 and 80 kDa in both human and mouse tissues with a tissue-specific variability of the isoform ratio. Spastin is an abundant protein in neural tissues and immunolabeling experiments have shown that spastin is expressed in neurons but not in glial cells. These data indicate that axonal degeneration linked to spastin mutations is caused by a primary defect of neurons. Protein and transcript analyses of patients carrying either nonsense or frameshift spastin mutations revealed neither truncated protein nor mutated transcripts, providing evidence that these mutations are responsible for a loss of spastin function. Identifying agents able to induce the expression of the non-mutated spastin allele should represent an attractive therapeutic strategy in this disease.

Early onset autosomal dominant spastic paraplegia caused by novel mutations in SPG3A.

Hereditary spastic paraplegia (HSP) is a group of neurodegenerative disorders mainly characterized by progressive spasticity of the lower limbs. The major features of HSP are a marked phenotypic variability both among and within families and an extended genetic heterogeneity. More than 20 HSP loci and 10 spastic paraplegia genes (SPG) have been identified to date, including the genes responsible for the two most frequent forms of autosomal dominant spastic paraplegia (AD-HSP), encoding spastin (SPG4) and atlastin (SPG3A), respectively. To date, only eight mutations have been described in the atlastin gene, which was reported to account for about 10% of all AD-HSP families. We investigated 15 German and French AD-HSP families, including the 3 large pedigrees that allowed the mapping and subsequent refinement of the SPG3A locus. Three novel mutations were found in exons 4, 9, and 12 of the atlastin gene and the common R239C mutation located in exon 7 was confirmed in a 7th family of European origin. Overall, the comparison of the clinical data for all SPG3A-HSP families reported to date failed to reveal any genotype/phenotype correlation as demonstrated for other forms of AD-HSP. However, it confirmed the early onset of this form of HSP, which was observed in almost all affected individuals with a mutation in the atlastin gene.

The DNA sequence and analysis of human chromosome 14.

Chromosome 14 is one of five acrocentric chromosomes in the human genome. These chromosomes are characterized by a heterochromatic short arm that contains essentially ribosomal RNA genes, and a euchromatic long arm in which most, if not all, of the protein-coding genes are located. The finished sequence of human chromosome 14 comprises 87,410,661 base pairs, representing 100% of its euchromatic portion, in a single continuous segment covering the entire long arm with no gaps. Two loci of crucial importance for the immune system, as well as more than 60 disease genes, have been localized so far on chromosome 14. We identified 1,050 genes and gene fragments, and 393 pseudogenes. On the basis of comparisons with other vertebrate genomes, we estimate that more than 96% of the chromosome 14 genes have been annotated. From an analysis of the CpG island occurrences, we estimate that 70% of these annotated genes are complete at their 5' end.