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The transport systems for proline and potassium represent two of the active transport systems in Escherichia coli. They have further similarities that their transport may be utilized as a response to osmotic perturbations in the environment. The exact mechanism of transport had not been totally elucidated. The transport of proline had been assumed to operate as a proton symport and as such had been used as a model system when other transport systems were being investigated. This study has demonstrated that the major route of proline uptake through the proline permease 1 (PP1), operates as a Na+ - proline cotransport which may accept Li+ in the place of Na+. Unusually, Na+ stimulates the Vmax of transport with little or no effect on the Km. In addition to this transport system, there are two other proline uptake systems which function primarily for the transport of betaine. The transport of K + is also facilitated by more than one system. The Kdp system is a K+ transporting ATPase; the TrkF system is a low rate transport system which may represent leak through another pathway. The TrkA transport system is the major system but the mechanism is not known. Transport through the system is energised by ATP and a pmf, while exchange through the system requires only ATP. The role of ATP was investigated in this study by the use of metabolic inhibitors and vesicles. It was determined that the availability of ATP affected the steady state level of potassium in the cells rather than the rate of potassium upake. It was speculated that ATP would act as a regulator of the system which would be driven by the pmf. ATP may regulate TrkA through phosphorylation or by allosteric modification of the carrier.

In wetland soils, hydrology is considered to be one of the primary factors shaping wetland function and microbial community structure, but plant-soil interactions are also important mechanisms affecting microbial nutrient transformations. The research presented here considered the interactive effect to describe how hydrology and the presence of plants alter the soil profile, the development of the bacterial community, and their associated functions. To achieve this goal, plots were established in three hydrologically-distinct regimes (Wet, Intermediate, and Dry) within a non-tidal freshwater wetland along the James River (Charles City County, Virginia). Inside each main plot, ten subplots were cleared of all aboveground plant material; five plots were left to re-grow (“Vegetated” reference), while the remaining five were weeded each week to maintain bare soil (“Clipped” treatment subplots). Manipulations were started at the beginning of the growing season, and sampling continued until the following winter. Every eight weeks, soil cores (30 cm) were collected and analyzed for a variety of soil properties (e.g., pH, OM, C:N, redox, vegetation and root biomass), microbial community structure (16S-rDNA-based T-RFLP),bacterial abundance (Acridine Orange Direct Count), and soil function (Extracellular Enzyme Activity (EEA)). A mixed-effects repeated measures analysis of variance (ANOVA) was used to better understand how each variable responded within each hydrological regime and treatment. Principal component analysis (PCA) and Partial Mantel tests were used to elucidate how saturation and vegetation influence the microbial community structure and soil enzyme function. Bacterial community properties and soil functions followed differences in soil saturation and associated physicochemical parameters (i.e., pH and redox). Correlations with wetland vegetation were primarily related to seasonal changes in plant community composition and biomass, and differences between experimental treatments were small. Evidence suggests the present plant species and the amount of above- and belowground biomass plays a more selective role shaping bacterial communities and soil function. Due to the short-term of this study and tight soil correlations, it is difficult to determine if observed differences are a product of the plant community or soil saturation, but it is clear that each is important. Based on the literature, plant effects were smaller in this wetland than might be expected. This experiment took place in a recently exposed lake basin, so plant-soil-microbe interaction may not be well established. As the wetland matures, relative importance of vegetation is expected to increase and impact bacterial composition and function. Collectively, these results demonstrate that wetlands are not a product of one separate variable, but result from various factors interlinked to shape microbial communities and soil functions.

Intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs) exist as interconverting conformational ensembles, without a single fixed three-dimensional structure in vivo. The focus in the literature up to this point has been primarily on IDPs that are mostly or entirely disordered. Therefore, we have an incomplete understanding of the incidence and functional relevance of IDPRs in proteins that have regions of both order and disorder. This work explores these populations, by examining IDPRs in the Protein Data Bank (PDB) and in enzymes. By applying disorder prediction methods combined with an analysis of missing regions in crystal structure data, this work shows that enzymes have a similar incidence and length of IDPRs as do non-enzymes, and that these IDPRs are correlated with functions related to macromolecular metabolism, signaling, and regulation. Furthermore, extensive analyses of missing regions with conflicting information between multiple structures in the PDB show that, rather than experimental artifacts, this ambiguity most likely arises due to partially or conditionally disordered regions. This work documents the first proteome level study of protein intrinsic disorder in enzyme populations and demonstrates a novel way of analyzing missing regions in the PDB. Furthermore, an extensive literature search as part of this work provides information for 1127 IDPs with experimental evidence documented in the literature, 96 of which are enzymes. The results contained herein present a new model of the protein universe, where disorder is directed by evolution in both non-enzymes and enzymes to make the most of limited proteomes in complex organisms through complicated signaling networks and tightly controlled regulation.

Flavonoids are an important group of secondary metabolites found in plants and have a wide variety of properties. Some play a role in fl ower pigmentation, while others have antimicrobial properties. Glucosylation is an important modifi cation of fl avonoids and is mediated by glucosyltransferases. In this process, the enzyme transfers glucose from UDP-glucose to a specifi c position on the fl avonoid. Previous study from the lab characterized a glucosyltransferase from C. paradisi that is fl avonol specifi c. In this study an attempt has been made to study the structure and function of this fl avonol specifi c glucosyltransferase using site directed mutagenesis. The glutamine residue at position 87 of the Cp-3-O-GT enzyme was changed to isoleucine, the analogous residue in the 3-O-glucosyltransferase of Clitoria ternatea. Similarly, the histidine at position 154 was changed to tyrosine. We hypothesize that these mutations will change substrate specifi city. The glutamate at position 88 was changed to an aspartic acid. We hypothesize that this will change the regiospecifi city of the enzyme, as aspartic acid is the analogous residue found in some 7-O-glucosyltransferases. Finally, we introduced a double mutation with glutamine 87 becoming isoleucine and glutamate 88 becoming aspartic acid, with the hypothesis that both regiospecifi city and substrate specifi city will be changed.

Tracking the evolution of function in enzyme superfamilies is key in understanding how important biological functions and mechanisms have evolved. New genes are being sequenced at a rate that far surpasses the ability of characterization by wet-lab techniques. Moreover, bioinformatics allows for the use of methods not amenable to wet lab experimentation. We now face a situation in which we are aware of the existence of many gene families but are ignorant of what they do and how they function. Even for families with many structurally and functionally characterized members, the prediction of function of ancestral sequences can be used to elucidate past patterns of evolution and highlight likely future trajectories. In this thesis, we apply in silico structure and function methods to predict the functions of protein sequences from two diverse superfamily case studies. In the first, the metallo-β-lactamase superfamily, many members have been structurally and functionally characterised. In this work, we asked how many times the same function has independently evolved in the same superfamily using ancestral sequence reconstruction, homology modelling and alignment to catalytic templates. We found that in only 5% of evolutionary scenarios assessed, was there evidence of a lactam hydrolysing ancestor. This could be taken as strong evidence that metallo-β-lactamase function has evolved independently on multiple occasions. This finding has important implications for predicting the evolution of antibiotic resistance in this protein fold. However, as discussed, the interpretation of this statistic is not clear-cut. In the second case study, we analysed protein sequences of the DUF-62 superfamily. In contrast to the metallo-β-lactmase superfamily, very few members of this superfamily have been structurally and functionally characterised. We used the analysis of alignment, gene context, species tree reconciliation and comparison of the rates of evolution to ask if other functions or cellular roles might exist in this family other than the ones already established. We find that multiple lines of evidence present a compelling case for the evolution of different functions within the Archaea, and propose possible cellular interactions and roles for members of this enzyme family.