64 research outputs found
Synthetic Studies Toward Complex Polycyclic Natural Products
The first chapter of this dissertation describes the use of an intramolecular Staudinger/aza-Wittig reaction in the synthesis of 1,2,5,6-tetrahydro-1,2,4-triazines, a structural motif of the natural product noelaquinone. The DEF ring system of noelaquinone was prepared in 13 steps and 2% overall yield with key steps featuring a Cu(I)-catalyzed C-arylation and the controlled acidic hydrolysis of the PMB protective group.
The second chapter describes the investigation of reactions between methyl 3-oxo-2-oxabicyclo[2.2.0]hexane-6-carboxylate and an indolo-indoline dimer in the presence of BF3·OEt2. Two tricyclic-fused heterocyclic products and a diene carboxylic acid have been obtained through a ring opening process, a retro-[2+2] cycloaddition, and a conjugate addition from the indole fragment.
The third chapter describes progress toward the total synthesis of haouamine A. Several routes to the marine alkaloid have been attempted. The challenges associated with the late stage lactam reduction, epoxidation, and aromatization strategy to prepare the necessary tetrahydropyridine and aza-cyclophane moieties are discussed
Construction of a Food-grade Expression Vector Based on pyrF Gene in Lactococcus lactis
In this study, the pyrF screening marker and the genomic DNA fragments were used to construct the expression vectors in food-grade Lactococcus lactis (L. lactis). Such expression system could potentially be used to express and produce food-grade and medicinal polypeptides. Firstly, the NZ3900 ÎpyrF auxotrophic strain was created from the homologous recombination mutant cassette. Secondly, the repA and repC genes were used as the replication elements, the pyrF gene as the screening marker, the P32 and P8 elements from L. lactis as the promoters, and the Tusp45 and TpepN from L. lactis as the terminators, all of which were constructed in the expression plasmid pLD. Finally, the reporter gene ZsGreen (a fluorescent protein) was used to verify the expression of recombinant protein in the NZ3900 ÎpyrF mutant strain and the genetic stability of pLD-ZsG plasmid. The result showed that the prototrophic ZsGreen positive transformants could grow normally in common Elliker culture medium, and the green fluorescent signal was observed under a fluorescence microscope. In addition, ZsGreen protein could be highly expressed in NZ3900 ÎpyrF and the expression plasmid could be stably transmitted through at least 30 generations, according to the results of the PCR and Western blotting, indicating that the recombinant protein was expressed in L. lactis in a stable manner. Based on the above results, the approach for creating an L. lactis expression vector (without antibiotic resistance gene) based on the pyrF auxotrophic marker is feasible and offers a basis for further investigation into the use of L. lactis to manufacture food- and pharmaceutical-grade polypeptides
Phage Encoded H-NS: A Potential Achilles Heel in the Bacterial Defence System
The relationship between phage and their microbial hosts is difficult to elucidate in complex natural ecosystems. Engineered systems performing enhanced biological phosphorus removal (EBPR), offer stable, lower complexity communities for studying phage-host interactions. Here, metagenomic data from an EBPR reactor dominated by Candidatus Accumulibacter phosphatis (CAP), led to the recovery of three complete and six partial phage genomes. Heat-stable nucleoid structuring (H-NS) protein, a global transcriptional repressor in bacteria, was identified in one of the complete phage genomes (EPV1), and was most similar to a homolog in CAP. We infer that EPV1 is a CAP-specific phage and has the potential to repress up to 6% of host genes based on the presence of putative H-NS binding sites in the CAP genome. These genes include CRISPR associated proteins and a Type III restriction-modification system, which are key host defense mechanisms against phage infection. Further, EPV1 was the only member of the phage community found in an EBPR microbial metagenome collected seven months prior. We propose that EPV1 laterally acquired H-NS from CAP providing it with a means to reduce bacterial defenses, a selective advantage over other phage in the EBPR system. Phage encoded H-NS could constitute a previously unrecognized weapon in the phage-host arms race
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Validation of two ribosomal RNA removal methods for microbial metatranscriptomics
Comparative Genomic Analysis of Neutrophilic Iron(II) Oxidizer Genomes for Candidate Genes in Extracellular Electron Transfer
Extracellular electron transfer (EET) is recognized as a key biochemical process in circumneutral pH Fe(II)-oxidizing bacteria (FeOB). In this study, we searched for candidate EET genes in 73 neutrophilic FeOB genomes, among which 43 genomes are complete or close-to-complete and the rest have estimated genome completeness ranging from 5 to 91%. These neutrophilic FeOB span members of the microaerophilic, anaerobic phototrophic, and anaerobic nitrate-reducing FeOB groups. We found that many microaerophilic and several anaerobic FeOB possess homologs of Cyc2, an outer membrane cytochrome c originally identified in Acidithiobacillus ferrooxidans. The âporin-cytochrome c complexâ (PCC) gene clusters homologous to MtoAB/PioAB are present in eight FeOB, accounting for 19% of complete and close-to-complete genomes examined, whereas PCC genes homologous to OmbB-OmaB-OmcB in Geobacter sulfurreducens are absent. Further, we discovered gene clusters that may potentially encode two novel PCC types. First, a cluster (tentatively named âPCC3â) encodes a porin, an extracellular and a periplasmic cytochrome c with remarkably large numbers of heme-binding motifs. Second, a cluster (tentatively named âPCC4â) encodes a porin and three periplasmic multiheme cytochromes c. A conserved inner membrane protein (IMP) encoded in PCC3 and PCC4 gene clusters might be responsible for translocating electrons across the inner membrane. Other bacteria possessing PCC3 and PCC4 are mostly Proteobacteria isolated from environments with a potential niche for Fe(II) oxidation. In addition to cytochrome c, multicopper oxidase (MCO) genes potentially involved in Fe(II) oxidation were also identified. Notably, candidate EET genes were not found in some FeOB, especially the anaerobic ones, probably suggesting EET genes or Fe(II) oxidation mechanisms are different from the searched models. Overall, based on current EET models, the search extends our understanding of bacterial EET and provides candidate genes for future research
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Microbial diversity in restored wetlands of San Francisco Bay
Wetland ecosystems may serve as either a source or a sink for atmospheric carbon andgreenhouse gases. This delicate carbon balance is influenced by the activity of belowgroundmicrobial communities that return carbon dioxide and methane to theatmosphere. Wetland restoration efforts in the San Francisco Bay-Delta region may helpto reverse land subsidence and possibly increase carbon storage in soils. However, theeffects of wetland restoration on microbial communities, which mediate soil metabolicactivity and carbon cycling, are poorly studied. In an effort to better understand theunderlying factors which shape the balance of carbon flux in wetland soils, we targetedthe microbial communities in a suite of restored and historic wetlands in the SanFrancisco Bay-Delta region. Using DNA and RNA sequencing, coupled with greenhousegas monitoring, we profiled the diversity and metabolic potential of the wetland soilmicrobial communities along biogeochemical and wetland age gradients. Our resultsshow relationships among geochemical gradients, availability of electron acceptors, andmicrobial community composition. Our study provides the first genomic glimpse intomicrobial populations in natural and restored wetlands of the San Francisco Bay-Deltaregion and provides a valuable benchmark for future studies
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Microbial diversity and carbon cycling in San Francisco Bay wetlands
Wetland restoration efforts in San Francisco Bay aim to rebuild habitat for endangeredspecies and provide an effective carbon storage solution, reversing land subsidencecaused by a century of industrial and agricultural development. However, the benefits ofcarbon sequestration may be negated by increased methane production in newlyconstructed wetlands, making these wetlands net greenhouse gas (GHG) sources to theatmosphere. We investigated the effects of wetland restoration on below-ground microbialcommunities responsible for GHG cycling in a suite of historic and restored wetlands in SF Bay. Using DNA and RNA sequencing, coupled with real-time GHG monitoring, we profiled the diversity and metabolic potential of wetland soil microbial communities. The wetland soils harbor diverse communities of bacteria and archaea whose membership varies with sampling location, proximity to plant roots and sampling depth. Our results also highlight the dramatic differences in GHG production between historic and restored wetlands and allow us to link microbial community composition and GHG cycling with key environmental variables including salinity, soil carbon and plant species
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Microbial diversity and carbon cycling in San Francisco Bay wetlands
Wetland restoration efforts in San Francisco Bay aim to rebuild habitat for endangeredspecies and provide an effective carbon storage solution, reversing land subsidencecaused by a century of industrial and agricultural development. However, the benefits ofcarbon sequestration may be negated by increased methane production in newlyconstructed wetlands, making these wetlands net greenhouse gas (GHG) sources to theatmosphere. We investigated the effects of wetland restoration on below-ground microbialcommunities responsible for GHG cycling in a suite of historic and restored wetlands in SF Bay. Using DNA and RNA sequencing, coupled with real-time GHG monitoring, we profiled the diversity and metabolic potential of wetland soil microbial communities. The wetland soils harbor diverse communities of bacteria and archaea whose membership varies with sampling location, proximity to plant roots and sampling depth. Our results also highlight the dramatic differences in GHG production between historic and restored wetlands and allow us to link microbial community composition and GHG cycling with key environmental variables including salinity, soil carbon and plant species
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