The Attwater’s prairie-chicken (Tympanuchus cupido attwateri; APC) is a species of grouse native to Texas coastal prairies and is on the critically endangered species list as a result of habitat destruction and overhunting. All of the current populations were captively bred and released into the wild. Survivorship for released APCs is very low, and individuals seldom survive to reproduce in the wild. One factor contributing to this may be an alteration in the gut microbiota as a result of captivity. Factors potentially influencing the gut microbial composition in captivity include antibiotic therapy, stress, and a predominantly commercially formulated diet. Recent studies have begun to shed light on the importance of the host microbial endosymbionts. Antibiotic administration, stress, diet, age, genotype and other factors have been shown to influence microbial populations in the gastrointestinal tracts of many different vertebrates. Sequencing of 16S rRNA gene amplicons on the Ion Torrent™ platform was used in this study to identify groups of bacteria in the cloacas as a surrogate for the gut microbiota in the APC. Antibiotic-treated and untreated birds, wild-hatched and captive-bred birds, and individuals sampled before and after release to the wild were examined. Significant differences were found between wild-hatched and captive raised birds both pre- and post release. In addition, there was extensive variation among the populations at the lower taxonomic ranks between individuals for each group of APCs. Principal coordinate analysis based on the weighted UniFrac distance metric further exhibited some clustering of individuals by treatment. These data suggest that captive breeding may have long-term effects on the cloacal microbiota of APCs with unknown consequences to their long-term health and survivorship.

Seventeen bacterial strains were isolated from 9 marine mud samples from the inshore environments of the East Sea. Four bacterial strains showed an inhibition against all tested microorganisms Staphylococcus aureus ATCC10832, Escherichia coli JM109, and Fusarium oxysporum. 16S rRNA sequences of four bacterial strains were obtained by PCR using specific primers. PCR products were cloned into E. coli DH5a using pJET1.2 blunt vector. The recombinant plasmids were sequenced and the lengths of these 16S rRNA sequences were ~930bp. The 16S rRNA sequence from the four bacterial DB1.2, DB1.2.3, DB4.2 and DB5.2 strain showed a high identity of 97 to 99% with the 16S rRNA sequence from Photobacterium sp., Oceanisphaera sp., Shigella sp., Stenotrophomonas sp, respectively. / Mười bảy chủng vi khuẩn đã được phân lập từ 9 mẫu bùn biển từ các vùng ven bờ biển Việt Nam. Bốn chủng vi khuẩn được ghi nhận có khả năng ức chế mạnh sự sinh trưởng và phát triển của các chủng vi khuẩn Staphylococcus aureus ATCC10832, Escherichia coli JM109, và thậm chí cả nấm Fusarium oxysporum. Trình tự gene 16S rRNA của bốn chủng vi khuẩn này đã được khuếch đại bằng PCR sử dụng cặp mồi đặc hiệu. Sản phẩm PCR được nối ghép vào vector pJET1.2 blunt sử dụng T4 ligase, hình thành plasmid tái tổ hợp và biến nạp vào E. coli DH5α. Khuẩn lạc có plasmid mang phân đoạn DNA chèn được nuôi cấy và tách plasmid. Trình tự 16S rRNA từ 4 chủng DB1.2, DB1.2.3, DB4.2 and DB5.2 chỉ ra có sự tương đồng 97 ÷ 99% so với trình tự 16S rRNA tương ứng của các chủng vi sinh vật biển trên ngân hàng gene thế giới là Photobacterium sp., Oceanisphaera sp., Shigella sp., và Stenotrophomonas sp.

The overall aim of this study was to investigate the potential influence of chitosan, a biodegradable and antimicrobial compound, on termite hindgut symbionts. For this purpose, a morphological quantifying technique was conducted on the protist community’s hindgut after feeding termites on chitosan-treated wood. The aim was to characterize the diversity of protist species in the economically important dark southern subterranean termite, Reticulitermes virginicus. A molecular phylogenetic analysis of the V3 and V4 hyper-variable regions of 16S ribosomal RNA (rRNA) gene of the bacterial community in the hindgut of R. virginicus was performed on termites exposed to chitosan treatment. Light microscopy visualization of protist species residing in the hindgut of workers showed presence of ten protist species both in the control sample and in termites fed a low concentration of chitosan. In this study, the coexistence of two species of the genus Trichonympha (T. agilis and T. burlesquei) is reported for the first time in R. virginicus. Monocercomonas sp. and Trichomitus trypanoides were the only two protists found in termites exposed to wood treated with higher chitosan concentration solutions and the absence of wood fragments in their food vacuoles was clear. This feature indicates that these two protists may not be involved in the digestion of the wood fragments impregnated with chitosan. The results of this study indicated that the potential effect of chitosan caused elimination of the protist species in termite hindguts. The genomic DNA of bacterial hindgut community of R. virginicus were profiled using sequences which amplified theV3-V4 sub-regions of 16S rRNA gene. Sequences were analyzed using a taxonomic analysis tool, Quantitative Insights Into Microbial Ecology (OIIME 2), in order to infer the effect of chitosan on the composition of the bacterial fauna in the hindgut. The richness and evenness results indicated that the most diversity was observed in the bacteria from termites not being exposed (UNX) to treatment compared to other treatment groups. On the other hand, the lowest richness and evenness were determined for chitosan-treated wood (CTE) and starved termites (STV). Of 28 bacterial phyla, Bacteroidetes, Firmicutes, Elusimicrobia, and Proteobacteria were the most dominant phyla across all the treatment groups. The results suggest that chitosan treated wood led to the microbial community shifts in R. virginicus. In addition, lack of a nutrition source and other changes in termite’s food affect the termite hindgut bacterial diversity.

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<p>Ribosome biogenesis is a major metabolic expense of bacteria and a promising target for antibacterial drug discovery. <em>Trans-</em>acting proteins, called ribosome biogenesis factors, aid this complex and cooperative process. EngA (YfgK, Der) is a widely distributed bacterial GTPase that is shown here to be important for normal ribosome biogenesis. EngA is an attractive antibacterial target because it is essential for viability in bacteria but is absent in humans.</p> <p>The GTPase activity and cellular function of EngA was investigated in <em>Escherichia coli</em>. Depletion of EngA caused accumulation of 30S and 50S ribosomal subunits at the expense of 70S ribosomes, showing for the first time that EngA is important for normal ribosome biogenesis. Mutation of either of the tandem GTPase domains of EngA led to abnormal ribosome profiles, cell death and loss of GTPase activity, revealing that the two GTPase domains act cooperatively to carry out an essential function. EngA bound the 50S subunit of the ribosome in cells and <em>in vitro</em>. Depletion of EngA resulted in sensitization to aminoglycoside antibiotics, which bind at the aminoacyl-tRNA binding site of ribosomes. To search for an inhibitor of ribosome biogenesis, a high-throughput screen of the GTPase activity of EngA was developed. A specific inhibitor was not identified, however, this robust screen can be extended to other compound libraries. Thus, we showed that the GTPase domains of EngA have a cooperative function in ribosome biogenesis, probably in maturation of the 50S subunit, and that EngA is an amenable target for further inhibitor screens.</p> / Doctor of Philosophy (PhD)

<p> Two studies are presented in this thesis. First part is about the origin of tRNA genes in Trypanosoma and Leishmania. These organisms have special mitochondrial DNA, termed kinetoplast DNA (kDNA), which is unique in its structure and function. kDNA is a massive network which is composed of thousands of connected DNA circles. Unlike most other mitochondrial genomes, there is no gene encoding tRNAs in their kDNAs. So all the tRNAs used in mitochondria must be encoded on nuclear genes and transported from the cytoplasm into the mitochondria. So our question of interest is where the tRNA genes in their nucleus come from. We carry out phylogenetic analysis of these genes and the corresponding ones in bacteria, mitochondria and eukaryotic nuclei. There is no evidence indicating gene transfer from mitochondria to nucleus on the basis of this analysis. These results are consistent with the simplest hypothesis, i.e. that all tRNA genes of Trypanosoma and Leishmania have the same origin as nuclear genes of other eukaryotes.</p> <p> The second part is about the comparison of eukaryote phylogenies obtained from mitochondrial rRNA and protein sequences. We carried out phylogenetic analysis for the species which have complete mitochondrial genomes by using both concatenated mitochondrial rRNA and protein sequences. We got phylogenies for three groups, fungi/metazoan, plant/algae and stramenopile/alveolate group. The analysis is useful for the further study of position of the genetic code changes and the mechanisms involved.</p> / Thesis / Master of Science (MSc)

Nitrification is the biological process whereby ammonia is converted first to nitrite by ammonia-oxidizing bacteria, and then the nitrite is subsequently converted to nitrate by nitrite-oxidizing bacteria. Ammonia and nitrite levels are closely monitored during treatment of wastewater due to their toxicity to other biological processes. Sybron Chemicals, Inc., is a company that manufactures a nitrifying bioaugmentation culture (1010N) that is used to enhance the naturally occurring levels of biological nitrification. The microbial population of the 1010N product has been examined using a combination of conventional bacteriological methods and modern molecular techniques, with the goal of developing nucleic acid probes that can be used to detect the product in an environmental sample. Small regions of the 16S rRNA genes of the bacteria in 1010N (and two new nitrifying enrichment cultures) were amplified via the polymerase chain reaction (PCR) and analyzed via temperature gradient gel electrophoresis (TGGE). TGGE is a procedure that allows for separation and visualization of individual PCR products that are the same size, based on differences in their sequence. Two of the predominant PCR products in 1010N were purified from the TGGE gel matrix, reamplified via PCR, and sequenced to allow for phylogenetic analysis and nucleic acid probe design. Coincidentally, two strains (NS500-9 and MPN2) that had been isolated from the 1010N mixed consortium and grown in pure culture were found, via TGGE, to have identical 16S rRNA sequences to the PCR products under investigation. Nearly the full-length 16S rRNA genes from these two organisms were PCR amplified, cloned, and sequenced in order to provide a basis for more accurate phylogenetic analysis. The two dominant organisms in the 1010N product, NS500-9 and MPN2, were thereby found to be most closely related to Nitrosomonas and Nitrobacter, respectively, in the existing database. Using the nucleic acid sequences of the cloned DNA, organism-specific DNA probes were designed for both NS500-9 and MPN2. Unfortunately, difficulties were encountered in using the probes to monitor 1010N activity levels via quantitative dot blot hybridizations (rRNA-DNA). Therefore, efforts were redirected to using the TGGE semi-quantitatively with an internal PCR standard (Brüggeman, et al., 2000) to estimate original cell numbers of 1010N within a mixed consortium. This method was not applicable to our system due to substantial preferential binding of the primers to template other than the standard. Samples from a laboratory-scale bioreactor, bioaugmented with 1010N, were used in an attempt to correlate an increase in activity with a detectable shift in population via TGGE. No detectable shift in population was detected in these samples even though the system exhibited increased levels of nitrification. Therefore, the sensitivity of the TGGE system was also examined by determining the limits of detection when 1010N was present in activated sludge. In both whole cell spiking experiments and genomic DNA spiking experiments, it was found that 1010N must be present at a level of at least 5% of the total population in order to be detected. While this provides some information about microbial populations, in order to evaluate the biological activity of a system, nucleic acid probes should be used in a rRNA based study. / Master of Science