Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009. / Committee Chair: Alfred H. Merrill, Jr; Committee Member: John Cairney; Committee Member: M. Cameron Sullards; Committee Member: Marion B. Sewer; Committee Member: Yuhong Fan. Part of the SMARTech Electronic Thesis and Dissertation Collection.

The ATP-PRTase enzyme catalyses the first committed step of histidine biosynthesis in archaea, bacteria, fungi and plants.1 As the catalyst of an energetically expensive pathway, ATP-PRTase is subject to a sophisticated, multilevel regulatory system.2 There are two families of this enzyme, the long form (HisGL) and the short form (HisGS) that differ in their molecular architecture. A single HisGL chain comprises three domains. Domains I and II house the active site of HisGL while domain III, a regulatory domain, forms the binding site for histidine as an allosteric inhibitor. The long form ATP-PRTase adopts a homo-hexameric quaternary structure.3,4 HisGS comprises a similar catalytic core to HisGL but is devoid of the regulatory domain and associates with a second protein, HisZ, to form a hetero-octameric assembly.5 This thesis explores the allosteric regulation of the short form ATP-PRTase, as well as the functional and evolutionary relationship between the two families. New insight into the mode allosteric inhibition of the short form ATP-PRTase from Lactococcus lactis is reported in chapter two. A conformational change upon histidine binding was revealed by small angle X-ray scattering, illuminating a potential mechanism for the allosteric inhibition of the enzyme. Additionally, characterisation of histidine binding to HisZ by isothermal titration calorimetry, in the presence and absence of HisGS, provided evidence toward the location of the functional allosteric binding site within the HisZ subunit. Chapter three details the extensive effort towards the purification of the short form ATP-PRTase from Neisseria menigitidis, the causative agent of bacterial meningitis. This enzyme is of particular interest as a potential target for novel, potent inhibitors to combat this disease. The attempts to purify the long form ATP-PRTase from E. coli, in order to clarify earlier research on the functional multimeric state of the enzyme, are also discussed. Chapter four reports the investigation of a third ATP-PRTase sequence architecture, in which hisZ and hisGS comprise a single open reading frame, forming a putative fusion enzyme. The engineering of two covalent linkers between HisZ and HisGS from L. lactis and the transfer of the regulatory domain from HisGL to HisGS, is also discussed, in an attempt to delineate the evolutionary pathway of the ATP-PRTase enzymes. Finally, the in vivo activity of each functional and putative ATP-PRTase was assessed by E. coli BW25113∆hisG complementation assays.

Most gram-negative organisms require lipopolysaccharide and its membrane anchor, lipid A, for growth and survival. Also known as endotoxin, lipid A is synthesized via a nine-step enzymatic process, culminating in a conserved hexa-acylated, bis-phosphorylated disaccharide of glucosamine. This framework is often altered by condition- or species-specific lipid A modifications, which change the biochemical properties of the molecule in response to and to defend against environmental stress signals. Here, we expound on two stories in different gram-negative organisms, both involving novel or unanticipated biochemistry that impacts lipid A structure. First, the missing acyltransferase in the Epsilonproteobacterium Helicobacter pylori lipid A biosynthesis pathway is identified. This enzyme transfers a secondary acyl chain to the 3'-linked primary acyl chain of lipid A like E. coli LpxM, but shares almost no sequence similarity with the E. coli acyltransferase. It is reannotated as LpxJ and demonstrated to possess an unprecedented ability to act before the 2'-secondary acyltransferase, LpxL, as well as the 3-deoxy-D-manno-octulosonic acid transferase, KdtA. LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E. coli LpxM homolog, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homolog. The second story focuses on regulation of modifications to endotoxin structure that occur after the conserved biosynthesis pathway. E. coli pmrD is shown to be required for PmrAB-dependent lipid A modifications in conditions that exclusively activate PhoPQ; this result proves that PmrD connects PhoPQ and PmrAB despite previous reports that it is an inactive connector in this organism. Further, RNA sequencing and polymyxin B survival assays solidify the role of E. coli pmrD in influencing expression of pmrA and its target genes and promoting survival during exposure to cationic antimicrobial peptides. Notably, the presence of an unknown factor or system capable of activating pmrD to promote lipid A modification in the absence of the PhoPQ system is also revealed. In all, the findings presented here expand our understanding of alternative approaches to lipid A biosynthesis and the complex systems that regulate modifications of this dynamic molecule.

Previously our laboratory had identified a novel component of the Staphylococcus aureus regulatory network, an extracytoplasmic function ó factor, óS, involved in stress response and disease causation. Here we present additional characterization of óS, demonstrating a role for it in protection against DNA damage, cell wall disruption and interaction with components of the innate immune system. Promoter mapping reveals the existence of four unique sigS start sites, one of which appears to be subject to auto-regulation. Transcriptional profiling revealed that sigS expression remains low in a number of S. aureus wild-types, but is upregulated in the highly mutated strain RN4220. Further analysis demonstrates sigS expression is inducible upon exposure to a variety of chemical stressors that elicit DNA damage, including methyl methanesulfonate (MMS) and ciprofloxacin, as well as those that disrupt cell wall stability, such as ampicillin and oxacillin. Ex vivo transcriptional analysis reveals that significant expression of sigS can be induced upon phagocytosis by RAW 264.7 murine macrophage-like cells. Regulation of óS appears to be unique, as the downstream encoded protein, SACOL1828, seemingly acts as a positive activator, rather than as an expected anti-sigma factor. Using a global transposon screen we have elucidated additional genes implicated in the regulation of sigS, including those involved in cell wall stability, cellular detoxification, virulence and DNA base excision repair. Phenotypically, óS mutants display sensitivity to a broad range of DNA damaging agents, such as ultraviolet light, MMS and ethidium bromide. These effects are seemingly mediated via regulation of the purine biosynthesis pathway, as microarray, proteomic and qRT-PCR analysis of óS mutants reveal decreased transcription of all genes involved. Enzymatic profiling of PurA involved in adenine biosynthesis, demonstrates decreased activity in the óS mutant. Finally, we provide further evidence for the role of óS in S. aureus pathogenesis, revealing that sigS mutants display decreased ability to cause localized infections and are impaired in their interactions with components of the human innate immune system. Collectively, our data argues for the important, and perhaps novel, role of óS in the stress and virulence responses of S. aureus.

Proteoglycans (PGs) are composed of highly sulfated glycosaminoglycans chains (GAGs) attached to specific core proteins. They are present in extracellular matrices, on the cell surface and in storage granules of hematopoietic cells. Heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS) GAGs play indispensable roles in a wide range of biological processes, where they can serve as protein carriers, be involved in growth factor or morphogen gradient formation and act as co-receptors in signaling processes. Protein binding abilities of GAGs are believed to be predominantly dependent on the arrangement of the sugar modifications, sulfation and epimerization, into specific oligosaccharide sequences. Although the process of HS and CS/DS assembly and modification is not fully understood, a set of GAG biosynthetic enzymes have been fairly well studied and several mutations in genes encoding for this Golgi machinery have been linked to human genetic disorders. This thesis focuses on the zebrafish N-deacetylase/N-sulfotransferase gene family, encoding key enzymes in HS chain modification, as well as glycosyltransferases responsible for chondroitin/dermatan sulfate elongation present in zebrafish. Our data illustrates the strict spatio-temporal expression of both the NDST enzymes (Paper I) and CS/DS glycosyltransferases (Paper II) in the developing zebrafish embryo. In Paper III we took advantage of the four preexisting zebrafish mutants with defective GAG biosynthesis. We could demonstrate a relation between HS content and the severity of the pectoral fin defects, and additionally correlate impaired HS biosynthesis with altered chondrocyte intercalation. Interestingly, altered CS biosynthesis resulted in loss of the chondrocyte extracellular matrix. One of the main findings was the demonstration of the ratio between the HS biosynthesis enzyme Extl3 and the Csgalnact1/Csgalnact2 proteins, as a main factor influencing the HS/CS ratio. In Paper IV we used the newly developed CRISPR/Cas9 technique to create a collection of zebrafish mutants with defective GAG biosynthetic machineries. Lack of phenotypes linked to null-mutations of most of the investigated genes is striking in this study.

Type II polyketide synthases (PKSs) are responsible for the generation of structurally diverse and clinically important aromatic polyketides. The bacteriostatic agent enterocin (enc) isolated from the marine microbe "Streptomyces maritimus" is derived from a rare benzoate primer unit and contains a unique nonaromatic caged core structure resulting from a Favorskii-like carbon skeleton rearrangement. The apparent diversion between enterocin biosynthesis and all other type II PKS pathways offered the opportunity to discover novel enzymatic strategies that may be exploited to diversify the chemical structures of polyketides. A comprehensive biochemical analysis was performed in order to characterize the key steps in enterocin biosynthesis and finally to reconstitute the whole pathway in vitro using purified recombinant enzymes.A nonribosomal peptide synthetase (NRPS)-like priming mechanism was discovered for the selective activation of a benzoic acid starter unit and its subsequent attachment to the enc PKS to initiate polyketide biosynthesis. This is the first example of a type II PKS that employs an NPRS-like priming mechanism to utilize alternative non- acetate starter units. Secondly, the minimal enc PKS was reconstituted in vitro to give three novel acetate-primed metabolites that had never been identified by heterologous in vivo expression of recombinant enc PKS gene sets. The minimal enc PKS was then merged with the NRPS-like chain initiation module and the resulting multienzyme complex catalyzed the formation of benzoate-primed natural products wailupemycin F and wailupemycin G. Favorskii-like rearrangement of the nascent polyketide chain was replicated in vitro and the flavin-dependent oxygenase EncM was confirmed to be solely responsible for catalyzing this unprecedented rearrangement. Other biosynthetic steps in the late stage of the enc pathway were also replicated in vitro, including the methylation of desmethyl-5-deoxyenterocin to 5-deoxyenterocin and the hydroxylation of 5-deoxyenterocin to enterocin.Finally, the entire enc type II PKS pathway was successfully assembled in vitro using ten recombinant proteins and three commercial enzymes. Five enc-based natural products were generated from benzoic acid and malonyl-coenzyme A. This biochemical investigation on enterocin biosynthesis represents the first complete in vitro reconstitution of a type II PKS system and also provides an alternative strategy to create complex natural products by multienzyme synthesis.

No description available.

Utilization of iron by hemoglobin-producing cells is highly efficient. The acquisition of iron from plasma requires the binding of diferric transferrin (Tf) to its cognate receptor (Tf-R) on the erythroid cell membrane, followed by internalization of the Tf - Tf-R complexes via receptor-mediated endocytosis. Through a poorly understood mechanism, iron is targeted to mitochondria, the site of heme biosynthesis. We believe that a direct interaction between iron-containing endosomes and mitochondria is essential for iron transfer to mitochondria and its efficient incorporation into heme. / In order to illustrate the interaction between endosomes and mitochondria, we have employed flow cytometry. Flow cytometry analysis of reticulocytes (erythrocyte precursors which still synthesize hemoglobin) stained with fluorescent dyes specific to mitochondria and endosomes revealed three distinct populations: mitochondria, endosomes and a population labeled with both dyes. This double-labeled population suggests a population composed of endosomes associated with mitochondria. Using non-fluorescent diferric-Tf, we were able to remove the double population, leaving only the endosomal and the mitochondrial population. This finding has confirmed that the double population is the result of the interaction between the two organelles. / Additionally, we established a cell-free assay consisting of fluorescent mitochondria and endosomes isolated from erythroid cells. Using confocal microscopy, we demonstrated a colocalization between the two organelles. We repeated the assay using fluorescent mitochondria and endosomes isolated from HeLa spinner cells. Using the mitochondrial uncoupler CCCP, we were able to significantly reduce the colocalization between the two organelles, indicating that the interaction between the organelles is specific and that the mitochondrial potential is a requirement for organellar interaction. / Based on our results from flow cytometry and confocal microscopy, we conclude that a specific and direct interaction exists between the two organelles.

Inflammation of the central nervous system (CNS) is important in many human diseases, and is regulated by a multitude of factors, including the cytokine interferon-gamma (IFNgamma). The importance of IFNgamma is highlighted in experimental autoimmune encephalomyelitis (EAE), an animal model of CNS inflammation. Mice lacking IFNgamma show exaggerated disease, with a different pattern of chemokine expression than the wild-type. We administered IFNgamma to the CNS using intrathecal injection of a replication-defective adenoviral vector to ask about direct actions of IFNgamma on chemokine expression without the confounding factors present during CNS inflammation. AdIFNgamma induced expression of CXCL10 and CCL5, two chemokines strikingly absent in Ifng-/- EAE. Chemokine expression was not associated with inflammation, though when an infectious stimulus was administered, an influx of immune cells to the CNS was seen. Using AdIFNgamma to restore IFNgamma to Ifng-/- mice with EAE had a disease-limiting effect. We used vectors encoding CXCL10 or CCL5, to replace these chemokines which are absent during Ifng-/- EAE, attempting to modulate the disease into a form resembling that of the wild-type. AdCCL5 treatment showed a mild reduction in EAE severity in the Ifng-/-, though AdCXCL10 treatment had no effect. A principal inducer of IFNgamma is interleukin-18 (IL 18), and IFNgamma induces IL18-binding protein (IL18bp) which inhibits IL18, establishing a negative feedback loop. We found that ILl8bp expression is upregulated in wild-type mice with EAE, but not in the Ifng-/-, suggesting that the exaggerated disease of the Ifng -/- may be due in part to unrestrained actions of ILI8. Treatment with a vector encoding IL18bp (AdIL18bp) significantly inhibited EAE, without restricting immune cell entry to the CNS. Cytokine expression was shifted away from a pattern favouring Th17 development. AdIL18bp treatment inhibited EAE in Ifng-/- mice, indicating that IFNgamma was not required for this activity. We used a vector encoding M3, a chemokine-binding protein derived from MHV-68, to reduce EAE severity, showing the first use of a viral chemokine-binding protein in EAE.

Hypoxoside, a phenolic diglucoside, with a diarylpentane-type structure, is thought to be the medicinally active constituent of corm extracts of Hypoxis hemerocallidea Fisch. & Mey. which are reputed to alleviate the symptoms of prostate hypertrophy and urinary infections. The biosynthes is and production of this unique phytochemical were investigated in H. hemerocallidea using both in vivo and in vitro systems. It was found, in root-producing callus, that [l4]C-phenylalanine and [14]C-t-cinnamic acid were efficient precursors for hypoxoside in comparison to [14]C-sodium acetate and [14]C-acetyl coenzyme-A, which were not incorporated into the phenolic compound. Thus, at least one aryl moiety of hypoxoside was derived, via phenylalanine and t-cinnamic acid, from the shikimate pathway. The acetate pathway did not appear to be involved in the biosynthetic process. The data supports the hypothesis that the molecule is formed from two cinnamate units with the loss of a carbon atom, in opposition to the proposal that the molecule is derived from head-to-tail condensation of acetate units onto a propenylic moiety. Despite the structural similarities between hypoxoside and caffeic and p-coumaric acids, these two hydroxycinnamic acids were not efficient precursors for hypoxoside in vivo or in vitro. A number of reasons are put forward to explain this finding. It was found that the greatest concentration of hypoxoside was located in the corms of intact plants. The major biosynthetic site of the molecule was also found to be located in this organ. Since the roots did accumulate the phytochemical to a small extent, the biosynthetic potential of these organs has not been disregarded. That of the leaves has been, however. The report by PAGE (1984) that the upper region of the corm contained a greater con cent ration of hypoxoside than the lower portion, is substantiated in this study, where this region was found to be more biosynthetically active than the lower half. Light microscopic and electron microscopic studies revealed that starch storing cells, which accumulated phenolics in their vacuoles, contained seemingly synthetically active tubular endoplasmic reticulum in their cytoplasm. A greater number of these cells were concentrated in the upper region as opposed to the lower half of the corm. It is postulated that these cells are the site for biosynthesis and accumulation of hypoxoside. The shikimate pathway, from which the precursors for hypoxoside are derived, was found, through the exposure of intact plants to [14]C-carbon dioxide, to be located mainly in the leaves. It is postulated from the above study and one in which [14]C-phenylalanine, [14]C-t-cinnamic acid, [14]C-p-coumaric acid and [14]C-caffeic acid were applied to intact plants, that phenylalanine and/or cinnamic acid are the transported form of the shi kimate derivatives. p-Coumaric and caffeic acids, which are metabolically more stable, are envisaged to be the sequestering forms. The investigation of the seasonal production of hypoxoside revealed that most of the synthesis and accumulation occurred after the corms had broken winter dormancy and after the flush of leaf growth had slowed down. During dormancy the production of hypoxoside appeared to cease. The in vjtro studies, where the effects of light, temperature, nutrients, plant growth regulators and supply of potential precursors, on hypoxoside production by root-producing callus were investigated, indicate that this metabolite is not simply a "shunt" metabolite. A number of factors other than precursor availability enhanced, or reduced the jn vjtro production of this phytochemical. Furthermore, production of the phytochemical and growth were not always antagonistic. Hypoxoside, the biosynthesis of which requires a more thorough investigation, is, however, according to this investigation, a typical secondary metabolite in many respects. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1989.