Graduate Thesis Or Dissertation
 

Molecular characterization of bacterial populations implicated inthe anaerobic metabolism of toxic plant alkaloids from two different experimental and environmental sources

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  • There have been many studies that describe the protective degradation or metabolism of potentially harmful plant toxins, such as, mimosine from Leucaena leuconcephala, pyrrolizidine alkaloids from tansy ragwort (Senecio jacobaea), oxalate and some mycotoxins by rumen microbes. There are many cases of plant-related toxicoses suffered by ruminant animals where there are no microbes known to offer protection. Ergot alkaloids produced by the endophytic fungus Neotyphodium coenophialum have been shown to cause the syndromes of fescue foot and summer slump in animals grazing tall fescue (Festuca arundinacea) grass varieties that carry the endophyte. Economic losses due to these diseases have been estimated to be about $1 billion per year. For this reason, it is economically desirable to find one or more microbes that could be introduced into the rumen environment to protect animals from fescue toxicosis and pyrrolizidine alkaloid toxicosis. Studies that have found protective microbes focused on identification of those microbes using mainly phenotyping and enrichment or isolation techniques. In the studies presented here, two different experimental and environmental systems were examined. Culture-independent molecular biology techniques were used to take advantage of differences found between bacteria in the 16S small ribosomal subunit gene as a means of characterizing diversity. Analytical chemistry techniques were used to place diversity observations in the context of function. Microbes within a consortia culture derived from the rumen of a sheep were previously shown to metabolize pyrrolizidine alkaloids found in the invasive weed, tansy ragwort. Thin layer chromatography was used to monitor microbial activity. The bacterial enrichment was characterized by molecular cloning techniques and by the molecular fingerprinting technique of denaturing gradient gel electrophoresis (DGGE). Phylogenetic analysis of the enrichment revealed that the consortium is composed of no more than five putative bacterial species which associated to the Anaerovibrio, Desulfovibrio, Megasphaera, Prevotella, and Synergistes generas. The DGGE results were directly compared to the cloning data by amplifying eight phylogenetically representative clones by polymerase chain reaction (PCR) and analyzing them by DGGE. Direct DGGE analysis of the enrichment displayed greater 16S diversity than the clone library used in this study, suggesting that at least one of the organisms present in the enrichment comprises less than 1% of the total cell population. Earthworms of the species Eisenia fetida are commonly used in the practice of vermicomposting. This is the process by which worms are used in a closed bin or pit, to aid in the breakdown of manure and other agricultural waste in order to generate highly fertile soil from the worm's digestive waste products, or cast. The worms were used in a two-treatment vermicompost experiment. One treatment was given tall fescue seed assayed to contain over 10,000 parts per billion of the endophyte toxin ergovaline (E+ treatment). The other treatment was used as a control and was given tall fescue seed with ergovaline amounts that fell below analytical levels of detection (E- treatment). Many of the worms originally introduced into the E+ treatment died off before a population of worms established itself as a productive vermicompost system. The success of this population of worms may be due to the presence of beneficial or protective microbes within their digestive tract. Digestive tracts were dissected and collected from worms of each treatment in February of 2007. The guts were homogenized and anaerobically cultured with and without added ergovaline. The disappearance of ergovaline and the stereo-isomer ergovalinine from worm-gut cultures was monitored daily by high-performance liquid chromatography (HPLC). DNA was purified from the same gut homogenate samples used for incubation experiments. Cloning of the 16S gene was applied to determine the identity of the bacteria present in the treatments during the month of February. The computational program Mr. Bayes was used to group restriction-fragment length polymorphism (RFLP) patterns based on their uniqueness. The program DOTUR was used to estimate the level of diversity based on ribotype distances. With the aim of encompassing the fullest amount of represented sequence diversity, these data were used to specifically select 192 clones from each 960 clone library. Selected clones were analyzed by sequencing the 16S insert. Clone sequences from both treatments made associations to bacterial 16S reference sequences in the phyla Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Planctomycetes, Alpha, Beta, Gamma, Delta and Epsilon proteobacteria, Verrucomicrobia and unclassifed bacterial sequences. Four clones associated best with a plastid sequence derived from a green algae species. The global similarity between libraries indicated that the populations were not significantly different at the time of sampling. HPLC data showed no observable loss of ergovaline from incubations of E+ or E- worm treatment samples taken in February. Incubation experiments in December of 2006 using only E+ treatment worm guts had demonstrated the potential for microbial metabolism of ergovaline; however, no molecular work was done at that time. An integrative approach was taken to describe the function and bacterial composition of the two systems studied. The use of analytical chemistry techniques placed the observations of microbial diversity into context. Thin-layer chromatography was used to check that the ability of the pyrrolizidine alkaloid-degrading enrichment was still intact after being revived from long-term storage. This allowed for direct comparisons to earlier data published about the identity of microbes within these enrichments. HPLC data indicated that worm-gut homogenates characterized in February did not demonstrate the same metabolic activity as was observed during an incubation of gut material taken from E+ treatment worms in December. Since there was no previous molecular data to compare potential differences in bacterial diversity from these two time points, the molecular data collected in February will be used as a reference of the background population at a time when there was no metabolism of ergovaline. It is intended that the results of these studies will further the understanding of which microbes are involved in the metabolism of the particular compound of interest. In this way, steps may be made toward refining the culturing and propagation of desirable bacteria that may potentially serve as a treatment for ruminant animals challenged by the presence of either pyrrolizidine or ergot alkaloids in their diets.
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