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• W137201178 abstract "Plants producing the highly toxic compound fluoroacetate are a significant problem in Australia because they are consumed by ruminant livestock thus causing economic loss to graziers. There are no antidotes for fluoroacetate poisoning which makes the problem difficult to control. Graziers currently rely on attempts to eradicate these toxic plants. However, the impact of removing this native Australian vegetation is also of concern. Fencing off grazing land to protect livestock is generally not always practical on extensive grazing properties, and is otherwise time consuming and expensive. There is a clear need to explore alternative strategies to protect the livestock from fluoroacetate poisoning. Microorganisms play a vital role in the digestive system of ruminant livestock because they break down the plant material to produce nutrients for the host animal. Some compounds in plant material that would otherwise be toxic are also degraded by complex metabolic capacities of gut microorganisms. Some research has focused on manipulating microbial populations in the gut to complement the animals own metabolic functions. In the case of fluoroacetate poisoning, past research has shown that recombinant rumen bacteria containing a fluoroacetate-degrading gene from soil bacteria are able to protect livestock animals through gastrointestinal detoxification. However, concerns over the release of these organisms into the general environment have prevented the use of this technology. There are no published reports of attempts to ascertain whether naturally- occurring fluoroacetate-degrading microorganisms exist in the gastrointestinal systems of animals. Knowledge of the rumen microbial ecosystem is useful in designing novel approaches to increase commercial animal production. To find a solution to fluoroacetate poisoning in livestock, our research is looking at naturally occurring microorganisms that have the ability to degrade fluoroacetate. There are currently several known species of aerobic soil bacteria and fungi that can degrade fluoroacetate. Bacterial strains of Burkholderia sp. and Delftia acidovorans, and fungal strains of Fusarium sp. produce enzymes capable of degrading fluoroacetate .The close proximity of gut and soil microorganisms may permit exchange of genetic material between the two ecosystems, especially under selective pressure. Interestingly, some native Australian animals exposed to fluoroacetate-producing vegetation in their diet have a much higher level of tolerance compared to the same species in unexposed areas. This resistance cannot currently be explained by differences in systemic metabolism between animals. vii To the best of our knowledge, there is no published research on attempts to investigate whether fluoroacetate-degrading microorganisms exist in the digestive systems of animals. Development of a colorimetric colony screening assay for detecting defluorination by microorganisms. Few microorganisms have been isolated that can cleave carbon-fluorine bonds. In this study, I report on a colorimetric colony-screening assay to facilitate the isolation of microorganisms capable of defluorination. The assay is based on a colorimetric reaction between a metal-dye chelate, zirconium-xylenol orange, and fluoride ions released from a fluorinated substrate through microbial metabolism. Depolymerised zirconium reagent gave the greatest visual contrast for the presence of fluoride compared to more polymerised forms of zirconium reagent. The sensitivity of the assay was greatest when the molar ratio of depolymerised zirconium to xylenol orange was 1:2. Using depolymerised zirconium and xylenol orange (150 nM and 300 nM respectively), the assay could detect a fluoride application spot (5 mM) containing 50 nmoles of fluoride ions. Most media constituents were well tolerated by the assay, although phosphate ions (present as potassium dihydrogen phosphate) needed to be restricted to 0.1 g/l, and some proteins digests (yeast extract, casein, tryptone and casamino acids) to between 1 -5 g/l. A microbial enrichment culture growing on solidified media containing 20 mM fluoroacetate was screened using the assay, and defluorinating bacteria belonging to the Burkholderia genus isolated. This method is sensitive, rapid and reliable for detecting defluorination by microorganisms growing on solidified media. An ecological analysis of microorganisms that degrade fluoroacetate. An integrated approach of culture-based enrichments and molecular detections methods was used to survey previously explored and some unexplored ecosystems such as the digestive systems of animals for fluoroacetate–degrading microorganisms. Aerobic environments such as soils and fresh water pond sediments could degrade fluoroacetate in culture, but not marine sediments or estuary water. A number of bacteria showing identity to the Burkholderia genus (99%) were isolated and found to contain novel fluoroacetate dehalogenase gene sequences (80-98%). The gut digesta from various herbivorous animals could degrade fluoroacetate anaerobically. Five previously uncultured fluoroacetate-degrading bacteria, which belonged to the Synergistetes phylum, were isolated from the gut of cattle (Bos taurus/indicus), kangaroos (Macropus spp.) and an emu (Dromaius novaehollandiae). Fluoroacetate dehalogenase genes could not be detected in these bacteria, or in their respective enrichment cultures using degenerate PCR assays, which may suggest a different enzyme or mechanism is involved in the fluoroacetate degradation. This is the first study to isolate dehalogenating bacteria from a gut ecosystem. viii Characterisation of a novel rumen bacterium able to degrade fluoroacetate. Microbial dehalogenation of chlorinated compounds in anaerobic environments is well known, but degradation of fluorinated compounds under similar conditions has rarely been described. Here I report on the isolation of a bovine rumen microorganism that metabolises fluoroacetate under anaerobic conditions, and a series of experiments that examine the mode of degradation. The bacterium was identified by 16S rRNA gene sequence analysis as belonging to the phylum Synergistetes and was designated strain MFA1. To date, no other Synergistetes are known to dehalogenate organic compounds. Growth was stimulated by amino acids with greater quantities of amino acids metabolised in the presence of fluoroacetate but sugars were not fermented. Acetate, formate, propionate, isobutryate, isovalerate, ornithine and hydrogen were end-products of amino acid metabolism. Acetate was the primary end-product of fluoroacetate dehalogenation and the amount produced correlated with the stoichiometric release of fluoride. The dehalogenation reaction was confirmed by analysis of the culture medium using fluorine nuclear magnetic resonance (19F NMR) spectroscopy, but fluorinated intermediates were not detected during growth. Hydrogen and formate produced in situ were consumed during dehalogenation. Results on the growth characteristics of strain MFA1 associated with fluoroacetate metabolism indicated that the bacterium may gain energy via reductive dehalogenation. This is the first study to identify a bacterium that can anaerobically dehalogenate fluoroacetate. Structure, abundance and composition of Synergistetes phylum bacteria in gut ecosystems. The phylogeny of Synergistetes phylum bacteria and a specific cluster of fluoroacetate-degrading bacteria, also from this phylum, were investigated in 38 gut ecosystems from 13 animal species. 16S rRNA phylum-specific PCR assays targeting the Synergistetes phylum showed the presence of this group in all the gut ecosystems investigated. The phylogeny of 299 16 S rDNA sequences showed distinct clustering based on the host. The majority of sequences fell within novel operational taxonomic units (OTUs). 16S rDNA strain-specific PCR assays targeting known fluoroacetate-degrading Synergistetes phylum bacteria showed their presence in 8 ecosystems (21%) from 7 animal species (54%). The analysis of 41 16 rDNA sequences from these ecosystems showed a distinct phylogenetic cluster (2% divergence). The results indicate that Synergistetes phylum bacteria are widely distributed through gastrointestinal ecosystems and that the diversity is much larger than previously recognised. The 16S rDNA sequence of known fluoroacetate- degrading microorganisms appears to be present in various hosts, but is likely to be a minor component of any one ecosystem." @default.
• W137201178 created "2016-06-24" @default.
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• W137201178 date "2011-03-01" @default.
• W137201178 modified "2023-09-27" @default.
• W137201178 title "INVESTIGATIONS ON THE MICROBIAL DEGRADATION OF FLUOROACETATE" @default.
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