Cyanide is a well known toxicant that arises in the environment from both biological and industrial sources. Bacteria have evolved novel coping mechanisms for cyanide and function as principal agents in the biosphere for cyanide recycling. Some bacteria exhibit the unusual ability of growing on cyanide as the sole nitrogen source. One such organism is Pseudomonas fluorescens NCIMB 11764 (Pf11764) which employs a novel oxidative mechanism for detoxifying and assimilating cyanide. A unique complex of enzymes referred to as cyanide oxygenase (CNO) is responsible for this ability converting cyanide to ammonia which is then assimilated. Because one component of the four member CNO complex was previously shown to act on cyanide independent of the other members, its characterization was sought as a means of gaining a better understanding of the overall catalytic mechanism of the complex. Preliminary studies suggested that the enzyme belonged to a subset of nitrilase enzymes known as cyanide dihydratases (CynD), however, a cynD-like gene in Pf11764 could not be detected by PCR. Instead, a separate nitrilase (Nit) linked to cyanide metabolism was detected. The corresponding nit gene was shown to be one of a conserved set of nit genes traced to a unique cluster in bacteria …

The aspartate transcarbamoylase (ATCase) was purified from Burkholderia cepacia 25416. In the course of purification, three different ATCase activities appeared namely dodecameric 550 kDa holoenzyme, and two trimeric ATCases of 140 kDa (consists of 47 kDa PyrB subunits) and 120 kDa (consists of 40 kDa PyrB subunits) each. The 120 kDa PyrB polypeptide arose by specific cleavage of the PyrB polypeptide between Ser74 and Val75 creating an active polypeptide short by 74 amino acids. Both the 40 and 47 kDa polypeptides produced active trimers. To compare the enzyme activity of these trimers, an effector assay using nucleotides was performed. The 140 kDa trimer showed inhibition while the 120 kDa polypeptide showed less inhibition. To verify the composition of the pyrBC holoenzyme complex, B. cepacia dihydroorotase (DHOase, subunit size of 45 kDa) was purified by the pMAL protein fusion and purification system and holoenzyme reconstruction was performed using purified ATCase and DHOase. Both the 140 kDa and the 120 kDa trimers could produce holoenzymes of 550 kDa and 510 kDa, respectively. The reconstructed ATCase holoenzyme from cleaved ATCase showed better reconstruction compared to that from uncleaved ATCase in the conventional ATCase activity gel assay. To characterize the relationship between pyrimidine pathway …

Super-resolution 3D imaging was achieved using newly synthesized photoactivatable quantum dot (PAQ dot) probes. Quantum dots were modified with a novel quencher system to make them photoactivatable. The unique properties of these PAQ dots enable single-fluorophore localization in three dimensions using a confocal microscopy optical sectioning method. Myosin and tropomyosin of rabbit myofibrilar bundles were specifically labeled with the newly synthesized PAQ dot. A sufficient number of single quantum dots were photoactivated, localized and reduced to their centroid and then reconstructed to a super-resolution image. The acquired super-resolution image shows a lateral and an axial sub-diffraction resolution and demonstrates ultrafine striations with widths less than 70 nm that are not evident by conventional confocal microscopy. The striations appear to be related to nebulin thin filament binding protein. This newly developed imaging system is cutting edge for its high resolution and localization as well its simplicity and convenience.

Factor VII, the initiator of the extrinsic coagulation cascade, circulates in human plasma mainly in its zymogen form, Factor VII and in small amounts in its activated form, Factor VIIa. However, the mechanism of initial generation of Factor VIIa is not known despite intensive research using currently available model systems. Earlier findings suggested serine proteases Factor VII activating protease, and hepsin play a role in activating Factor VII, however, it has remained controversial. In this work I estimated the levels of Factor VIIa and Factor VII for the first time in adult zebrafish plasma and also reevaluated the role of the above two serine proteases in activating Factor VII in vivo using zebrafish as a model system. Knockdown of factor VII activating protease did not reduce Factor VIIa levels while hepsin knockdown reduced Factor VIIa levels. After identifying role of hepsin in Factor VII activation in zebrafish, I wanted to identify novel serine proteases playing a role in Factor VII activation. However, a large scale knockdown of all serine proteases in zebrafish genome using available knockdown techniques is prohibitively expensive. Hence, I developed an inexpensive gene knockdown method which was validated with IIb gene knockdown, and knockdown all serine proteases …

Brucella ovis is a sexually transmitted, facultatively anaerobic, intracellular bacterial pathogen of sheep (Ovis aries) and red deer (Cervus elaphus). Brucella spp. infect primarily by penetrating the mucosa and are phagocytized by host macrophages, where survival and replication occurs. At least in some species, it has been shown that entry into stationary phase is necessary for successful infection. Brucella, like other alphaproteobacteria, lack the canonical stationary phase sigma factor ?s. Research on diverse members of this large phylogenetic group indicate the widespread presence of a conserved four-gene set including an alternative ECF sigma factor, an anti-sigma factor, a response regulator (RR), and a histidine kinase (HK). The first description of the system was made in Methylobacterium extorquens where the RR, named PhyR, was found to regulate the sigma factor activity by sequestering the anti-sigma factor in a process termed "sigma factor mimicry." These systems have been associated with various types of extracellular stress responses in a number of environmental bacteria. I hypothesized that homologous genetic sequences (Bov_1604-1607), which are similarly found among all Brucella species, may regulate survival functions during pathogenesis. To further explore the involvement of this system to conditions analogous to those occurring during infection, pure cultures of …

Pseudomonas aeruginosa are medically important bacteria that are notorious for causing nosocomial infections. To gain more knowledge into understanding how this organism operates, it was decided to explore the pyrimidine biosynthetic pathway. Pyrimidine synthesis, being one half of the DNA structure, makes it a very important pathway to the organism’s survivability. With previous studies being done on various genes in the pathway, pyrE has not been studied to the fullest extent. To study the function of pyrE, a site directed mutagenesis was done to completely knock out pyrE, which encodes the protein orotate phosphoribosyl transferase that is responsible for converting orotate into orotate monophosphate (OMP). A mutation in this step leads to accumulation and secretion of orotate into the medium. Analyzing virulence factors produced by the mutant and comparing to the wild type, some intriguing features of the mutant were discovered. One of the findings was the over expression of virulence factors pyoverdin and pyocyanin. Pyocyanin over expression, based on the results of this study, is due to the accumulation of orotate while over production of pyoverdin is due to the accumulation of dihydroorotate. The other virulence factors studied were motility assays, exoproducts, and growth analysis. All virulence factor production …