The human cellular proto-oncogene c-myb has been implicated as important in the regulation of hematopoietic cell growth and differentiation. Aberrant expression of this gene and chromosomal aberrations near the c-myb locus have been associated with a number of carcinogenic processes. An alternatively spliced CDNA clone of c-myb, pMbm-2, contains unique 5’ sequences which replace exon 1. The existence of this 5’ divergent CDNA clone led us into a study of the promoter activity of the c-myb gene. Intron 1 of c-myb is highly conserved between human and mouse throughout the intron, while only those sequences directly adjacent to exons 1 and 2 are conserved between human and chicken. The unique sequence of pMbm-2 was located directly adjacent to exon 2, suggesting that it arose as a product of alternative transcription initiation within intron 1. A cluster of transcription start sites was detected at the 5’ end of exon 2. Levels of messages utilizing these start sites are expressed proportionally to those arising from the primary promoter. Functional characterization of this region revealed that this region can function as a promoter. Deletion studies have revealed the presence of negative and positive regulatory elements within this region which are utilized with different efficiencies in different cell lines. These studies suggest that cis or trans factors acting in this region may serve a dual function in both attenuation and transcription initiation. Studies of the c-myb promoter utilizing the acute lymphoblastic cell line CCRF-CEM revealed that a portion of the c-myb promoter is lost in this cell line. The rearranged locus, which we have designated MRR (myb rearranged region), has been Cloned and mapped to chromosome 6. The MR sequence is linked to the c-myb locus, suggesting that the rearrangement is due to a submicroscopic deletion. The rearrangement appears to have no effect on c-myb promoter activity as analyzed in CCRF-CEM cells. The normal locus of the MRR sequence shows a high degree of homology to a member of the myc family of oncogenes. Therefore, although attenuation may be the primary mechanism of c-myb regulation, the existence of a second promoter in the c-myb gene and a rearrangement of the primary c-myb promoter in a leukemia cell line suggests that other regions at the 5’ end of this gene are important in the regulation of c-myb transcription.

We have identified a late, committed stage in the differentiation of the mast cell progenitor just prior to granulation. This mast cell-committed progenitor (MCCP) differs from the more primitive bone marrow mast cell progenitor in that it is able to proliferate and differentiate in the absence of interleukin-3 (IL-3) when cultured on a monolayer of embryonic skin or 3T3 fibroblasts. The MCCP can be harvested from the mesenteric lymph nodes of mice in their fourteenth day of infection with the rodent hookworm Nippostrongylus brasiliensis and can be cloned in a methylcellulose culture system by supplementing the cultures with fibroblast-conditioned medium from monolayers of embryonic skin or 3T3 fibroblasts. The mesenteric lymph node was virtually uncontaminated with hematopoietic progenitors other than the MCCP. The MCCP acquires a mucosa! mast cell phenotype when cloned in the presence of IL-3, but begins to take on a connective tissue mast cell phenotype when cloned in the presence of fibroblast conditioned medium. Upon fractionation by size exclusion chromatography, fibroblast conditioned medium contained a novel protein which supported proliferation of the MCCP and a separate granulation factor; thereby, proliferation and granulation can be uncoupled in vitro. These data demonstrate that IL-3 independent proliferation and differentiation of the MCCP does not require cell contact with fibroblasts. T cell-depleted cultures consistently produced higher numbers of mast cells than did nondepleted cultures. IL-3 production in the mesenteric lymph node peaked at day 11 and may instrumental in the transition of the IL-3 dependent progenitor into the MCCP. When mast cell-deficient SI/SId or W/Wv mice were infected with Nippostrongylus, SI/SId mice, but not W/Wv' mice, produced the MCCP. To determine if these mice make the fibroblast derived factors that support development of the MCCP, monolayers were prepared from skin connective tissues of SI/SId and W/Wv mice, and MCCP from normal mice were cloned in the presence of conditioned medium from these monolayers. Fibroblast conditioned mediurn from monolayers prepared from W/Wv mice, but not SI/SId mice, was able to support the development of mast cell colonies from MCCP.

Conversion of biomass to useful bio-products is a lengthy and often inefficient process. Research has looked at the conversion of cellulose to ethanol by means of termite bacterial consortium in fluidized bed bioreactor, where the bacteria are attached as biofilm to an activated carbon or cellulose support. High conversion rates were achieved in this reactor and the process was fairly robust and flexible. However establishing the biofilm on the activated carbon was a lengthy process in terms of the time it took the bacteria to attach to the various substrates (Activated Carbon and Cellulose) and form flocculants. The formation of such flocculants substantially increases the reaction surface and hence should optimize the production of ethanol. Many physical, chemical, and biological interactions facilitate the attachment of bacteria to surfaces. It was in this study that the electrostatic attraction was investigated. The understanding of the physical modifications of surface charge was chosen to be investigated in order to understand the ideal conditions to propagate and increase biofilm creation. Bacteria carry a negative surface charge and hence for increased attraction, the surface charge of the substrate should be modified to be positive. This research, performed as batch processes, has shown that with the correct surface charge modifications of the substrate the electrostatic attraction forces between the surface and the bacteria are maximized. As a result of the strong electrostatic attraction forces between the two surfaces the bacteria adsorbs and attaches to the substrate quicker and creates a biofilm on the surface. Prior to the attachment investigation it was important to attempt to understand the bacteria consortium within the termite gut.. The “worker” termites collect the food and feed off the soil whilst building their mound.. The bacteria found within the termite are in line with cellulose degradation, which can be manipulated for biofuels production. This study aimed to investigate a series of procedures of charge manipulation to the surfaces of both the substrate and bacteria in order to see the influence of electrostatic interaction on biofilm creation. It was seen that when only the activated carbons surface charge was modified to have a positive net charge the attachment of bacteria was most prominent. In addition it was proven that after being charged to a pH of 5 (4.28 x 104 Bacteria/mm2 Activated Carbon), 6 (3.90 x 104 Optimization of Microbial Adsorption (Biofilm Creation) on Activated Carbon by Surface Modification of Substrate 2013 Bacteria/mm2 Activated Carbon) and 7 (1.58 x 105 Bacteria/mm2 Activated Carbon) were achieved. The greatest attachment was seen when the activated carbon was charged to a pH of 7. This can be explained as the optimal positive pH the activated carbon should be charged to if its pHPZC is 9.7. If the pH is dropped lower than 7 although the surface becomes more positive, the surface actually approaches the surface charge of bacteria and the electrostatic interaction dissipates. In an effort to use the optimal pH modification to create a biofilm on the surface of the activated carbon time experiments were performed. After 13 days biofilms on the surface of the activated carbon surfaces were seen. The bacteria attach to surface of the activated carbon and produce Extracellular Polymers Substance (EPS) fluid which then causes other bacteria to attach to them resulting in the formation of Biofilms.

The mechanism of prokaryotic transcription has been characterized primarily in the classic system, Escherichia coli, and cannot be confidently extended to include other prokaryotic species, such as those of the Actinobacteria phylum. Actinobacteria represents a diverse group of Gram-positive species that range from soil dwellers to obligate pathogens, such as Mycobacterium tuberculosis (Tb). These species encode RNA polymerase (RNAP) binding proteins that are not present in model organisms, and therefore present a unique lens through which the basic mechanism of transcription can be further explored outside of model systems. In addition, these mechanisms of transcriptional regulation can be studied in the context of M. Tuberculosis pathogenesis. The model we use for tuberculosis is Mycobacterium Smegmatis, a homologue, which has a faster doubling time and is only Biosafety level 1. Within Actinobacteria, notable conserved RNAP binding proteins include RNA polymerase binding protein A (RbpA) and CarD. RbpA is specific to Actinobacteria, binding the β subunit of RNAP and primary σ factors. CarD binds to the β subunit and associates with DNA. Both proteins are upregulated upon exposure to stress, and have implications in the initiation of rRNA transcription. Each is proposed to stimulate the formation of transcriptionally competent RNAP-holoenzyme open promoter complexes, and CarD is thought to act as a global transcriptional regulator. RbpA and CarD are believed to be essential in M. Tuberculosis and M. Smegmatis. Recent structural analyses of RbpA and CarD suggest the two proteins may share a region of similarity that could compete for binding to the β subunit, and brings into question whether the two proteins are capable of coordinately modulating transcription or antagonize each other's activity. This was investigated through purification of CarD and RbpA and in vitro studies performed with [α-32P] Uridine triphosphate used to measure the level of transcription. These experiments led to the conclusion that RbpA and CarD are able to associate with the same RNAP and have an additive stabilizing action on the polymerase. Whether or not RbpA is an essential protein was also investigated genetically, and by using a Tetracycline on/off system. Sigma factors play an important role in transcription due to their ability to recognize promoter regions and initiate transcription. One connection that we have preliminary data for, through DNA pull downs, is that sigF binds rRNA promoters, and CarD and RbpA are both studied in the context of rRNA transcription. Therefore sigF is another factor that could be regulating rRNA transcription, possibly during stress. SigF is also the sigma factor that responds to oxidative stress, and CarD is involved in oxidative stress. Sigma F is a member of a family of 13 different sigma factors that are preset in M. Tuberculosis. There are two different types of sigma factors: primary, which are essential for normal growth, and alternative, which are typically expressed during differing environmental conditions. Sigma F has been shown to be upregulated during oxidative stress, which is why it was of particular interest to us. To investigate the roles of sig F, we exposed sig F deletion mutants and wild type strains to oxidative stress and measured ribosomal RNA production by reverse transcription quantitative real time PCR. It was concluded that sigF is a probable suppressor of rRNA when exposed to oxidative stress.

No description available.

The opportunistic pathogen Pseudomonas aeruginosa has been well studied for its ability to cause nosocomial infections in immunocompromised patients. However, its pathogenicity is only one aspect of the biology that makes this bacterium one of the most versatile of its genus. Since its first description in 1885, P. aeruginosa has been known to produce colorful, small molecules called phenazines. These redox-active compounds were originally thought of as mere secondary metabolites or virulence factors that allow P. aeruginosa to infect plant and animal hosts. However, recently we have gained an appreciation for their diverse functions that directly benefit their producer: phenazines act as signaling molecules, regulate intracellular redox homeostasis and are implicated in iron uptake. As a result, phenazines also have dramatic effects on the structural development of multicellular communities of P. aeruginosa, generally referred to as biofilms. How phenazine production is regulated in response to environmental cues to allow for this functional diversity is still poorly understood. Pseudomonas aeruginosa produces at least five different phenazines, each of which have distinct chemical properties. The genes encoding the core phenazine biosynthetic enzymes are found in two redundant 7-gene operons. These operons, phzA1-G1 (phz1) and phzA2-G2 (phz2), encode two sets of proteins that catalyze the synthesis of phenazine-1-carboxylic acid (PCA), the precursor for all other phenazine derivatives. Although the phz1 and phz2 operons are nearly identical (~98% similarity), they are differentially regulated. phz1 is regulated by quorum sensing (QS), while the factors controlling phz2 expression have not yet been identified. Furthermore, the contribution of phz2 to phenazine production is not fully understood. The phz2 operon is conserved among all P. aeruginosa species and we hypothesize that it may be vital to their ability to adapt to diverse environments. In this work, we have investigated the regulation of the phz2 operon and its contribution to colony biofilm development in P. aeruginosa PA14 (Chapter 2). We found that (1) phenazine production in biofilms is mediated exclusively through the phz2 operon, (2) phz2 expression is required for biofilm development and host colonization and (3) phz2 is regulated by quinolones, which are prominent signaling molecules in P. aeruginosa's QS system. We then investigated the roles of individual phenazines in colony development (Chapter 3) and the specificity of SoxR activation by redox active molecules (Chapter 4). We found that the effects of individual phenazines are not redundant and may be used in combination to modulate colony development. SoxR is a transcription factor that is activated by redox-active molecules including phenazines. Investigations into SoxR specificity showed that SoxR activation in non-enteric bacteria is tuned to specific redox potentials. Together, the findings presented in this thesis have expanded our knowledge about the role of phenazine production in biofilms and pathogenicity.

The rate of HIV-1 replication has an impact on the viral loads patients have and the time it takes for an infection to progress to AIDS. This replication rate is defined partly by the time it takes an infected cell to begin producing new infectious virus, and this, in turn, is defined by the time required for each step of the viral life cycle inside cells. Many of the stages of the HIV-1 life cycle have been well-characterized mechanistically, but the timing with which they occur has not. HIV-1 is under strong pressure to replicate rapidly, yet evidence indicates that there are stages at which there is active viral auto-inhibition of the rate of replication. We therefore sought to characterize the timing of each major stage of the viral life cycle and to determine how they are correlated with one another. Using a variety of techniques including quantitative microscopy we tracked the timing of these events, both in bulk infected cultures and in single infected cells, and generated a time line of the HIV-1 replication cycle. We find that there is a delay of about 11 hours between integration and gene expression, whereas early and late gene expression are separated by only about 3 hours. In addition we find that a critical event prior to assembly, the virus-directed removal of the host restriction factor APOBEC3G, takes place within 2.5 hours following late gene expression. One of the major processes HIV-1 must complete before it can produce new virions is the clearance of antiviral restriction factors that can block the production of new infectious virus. We present evidence in support of the hypothesis that the assembly and release of virions, which is inhibited by the presence of the MA domain of the Gag protein, is delayed precisely in order to allow restriction factor clearance to reach completion before the assembly process begins.

Lactobacillus iners, is a gram positive organism recently identified through the use of culture independent techniques. Identified as a major constituent of the vaginal microbiota, epidemiological studies have suggested that this organism may not provide the protective effects ascribed to other vaginal lactobacilli. Our work here has identified and characterized a pore forming toxin, Inerolysin, produced by this organism. This pore forming toxin was present in all strains of L. iners tested and possessed characteristics which firmly categorize it as a member of the Cholesterol Dependent Cytolsin superfamily. Additionally, we identified pH as a regulatory factor for the activity of Inerolysin as well as other CDCs. Inerolysin was shown to had optimal activity at acidic pH, while other toxins such as pneumolysin and arcanolysin had optimal activity at basic pH. We demonstrate that pH induced changes in activity were reversible, suggesting that a reversible conformational change takes place in the protein. Furthermore, our results show that it is the last step in pore formation, the transition from pre-pore to pore, which is impaired. Our attempts to localize this to particular residues were unsuccessful. Finally, we sought to understand what the vaginal environment of a Lactobacillus dominated flora would look like. We demonstrate that L. iners induced unique signaling in the vaginal epithelium, leading to the production of a unique profile of proinflammatory cytokines as well as antimicrobial peptides. We further demonstrate that some of these responses are mediated by the activity of the pore forming toxin of L. iners, Inerolysin.

No description available.

A method for measuring microbial degradation and mineralization of radiolabeled native chitin is described. ('14)C-labeled chitin was synthesized in vivo by injecting shed blue crabs (Callinectes sapidus) with N-acetyl-D- ('14)C -glucosamine, allowing for its incorporation into the exoskeleton. The cuticle had a total organic content of 0.48 mg C mg('-1) with a specific radioactivity of 6762 DPM mg('-1). Glucosamine i.e. chitin content, as determined colorimetrically, was 22% (w/w). Rates of chitin degradation and mineralization in estuarine water and sediments were determined as functions of temperature, inoculum source, and oxygen condition. Significant differences in rates between temperature treatments were evident. Q(,10) values ranged from 1.2 to 2.5 for water and sediment, respectively. Increased incubation temperature also resulted in decreased lag times before onset of chitinoclastic bacterial growth and chitin degradation. The highest rate, 284 mg day('-1)g('-1) seeded chitin, occurred in the water column inoculum at 25�C. The lowest rate, 83 mg day('-1) g('-1), was found in the anaerobic mud inoculum incubated at 15�C. Over 95% of particulate chitin degraded by water column bacteria was mineralized to CO(,2) with no apparent lag between processes. No measurable dissolved pool of radiolabel was present. Conversely, only 75-80% of chitin degraded by sediment inocula was mineralized. Label recoveries in the dissolved pool ranged from 6 to 17%. The anaerobic treatment possessed the highest pool of dissolved organic carbon due to the fermentation of chitin to volatile fatty acids. The anaerobic pathway of chitin decomposition by chitinoclastic bacteria was examined with an emphasis on end product coupling to other bacterial types. Actively growing chitinoclastic bacterial isolates produced primarily acetate, hydrogen, and carbon dioxide in broth culture. No sulfate reducing or methanogenic isolates grew on chitin as sole carbon source nor produced any measureable degradation products. Mixed cultures of chitin degraders and sulfate reducers resulted in positive sulfide production. Mixed cultures of chitin degrading isolates with methanogens resulted in the production of methane with concurrent reductions in headspace hydrogen and carbon dioxide. The combination of all three metabolic types resulted in the simultaneous production of methane and sulfide. More methane was produced in mixed cultures containing CO(,2)-reducing methanogens and acetoclastic sulfate reducers due to less interspecific hydrogen competition.