No description available.

No description available.

modC is part of a set of genes, modABC, which encode the bacterial molybdate ABC transporter. ModC comprises the ATPase. Of the three structural Mod proteins, ModC has received the least attention, believed to be little different from ATPases of other ABC complexes. However, findings of multiple copies of modC in A. vinelandii and identification of a potential molybdate-binding domain in the C-terminal of ModC may point to a more complex role. ORF10 is one of three copies of modC in A. vinelandii; atypically, ORF10 is not found with genes encoding the other ABC transporter components but is instead found as part of the nif gene cluster, encoding molybdenum nitrogenase. The role of ORF10 is investigated here via sequence analysis and comparison of growth of a ORF10- A. vinelandii strain with wild type growth. Findings imply ORF10 optimizes growth under conditions requiring nitrogenixation, specifically under those conditions where molybdate-availability is limited.

No description available.

Aspergillus flavus is the most common causal agent of aflatoxin contamination of food and feed. However, aflatoxin-producing potential varies widely among A. flavus genotypes with many producing no aflatoxins. Some non-aflatoxigenic genotypes are used as biocontrol agents to prevent contamination. Aflatoxin biosynthesis genes are tightly clustered in a highly conserved order. Gene deletions and presence of single nucleotide polymorphisms (SNPs) in aflatoxin biosynthesis genes are often associated with A. flavus inability to produce aflatoxins. In order to identify mechanisms of non-aflatoxigenicity in non-aflatoxigenic genotypes of value in aflatoxin biocontrol, complete cluster sequences of 35 A. flavus genotypes from Africa and North America were analyzed. Inability of some genotypes to produce aflatoxin resulted from deletion of biosynthesis genes. In other genotypes, non-aflatoxigenicity originated from SNP formation. The process of degeneration differed across the gene cluster; genes involved in early biosynthesis stages were more likely to be deleted while genes involved in later stages displayed high frequencies of SNPs. Comparative analyses of aflatoxin gene clusters provides insight into the diversity of mechanisms of non-aflatoxigenicity in A. flavus genotypes used as biological control agents. The sequences provide resources for both diagnosis of non-aflatoxigenicity and monitoring of biocontrol genotypes during biopesticide manufacture and in the environment.

Staphylococcus aureus is the leading cause of infective endocarditis in the United States. Infective endocarditis (IE) is defined as an infection of the endocardium, typically involving the heart valves. The hallmark features of IE are vegetations. Vegetations are cauliflower-like, stratified biofilms of bacteria and host factors that develop on the valve leaflets of the heart. The mechanisms of how vegetations form are not well understood, and as a consequence the bacterial factors that are important for development of IE are not well defined. My studies focus on the role of a family of S. aureus exoproteins known as superantigens and their role in IE. Superantigens (SAgs) are a class of secreted virulence factors that have been extensively studied for their role in systemic diseases such as toxic shock syndrome (TSS), pneumonia, and food poisoning. The SAg protein family is comprised of 23 distinct members designated as staphylococcal enterotoxin (SE) or enterotoxin-like (SEl) and toxic shock syndrome toxin-1 (TSST-1). The term superantigen is derived from the ability of SAgs to interact with the immune system, resulting in a nearly 3000-fold increase in activation when compared to standard antigens. SAgs have a defined structure that is composed of 2 domains, a carboxy-terminal beta-grasp domain and amino-terminal oligosaccharide/oligonucleotide binding (OB) fold. Defined groups of SAgs are associated with S. aureus strains isolated from specific diseases, but few studies have been done to determine the role of SAgs in diseases outside of TSS and food poisoning. The enterotoxin gene cluster (egc) is a group of 6 SAgs (selo, selm, sei, selu, seln, and seg) assembled into an operon-like cluster that is present in the majority of S. aureus strains isolated from IE patients. My studies have determined that the egc is able to induce vegetations when expressed in avirulent S. aureus strains. This is the first time the egc has been directly associated with IE. I further characterized the capacity of the individual egc proteins to induce vegetations. Four (selo, selm, sei, and selu) of the 6 egc SAgs were able to induce vegetation formation. This is the first time the individual egc proteins have been characterized and directly associated with IE. I also demonstrated that the egc proteins may not be exclusively expressed as a single polycistronic transcript but that selu and seg contain promoter elements that may drive their individual expression. Lastly, I provide evidence that the egc SAgs may be regulated by MgrA, a global regulator of S. aureus associated with virulence factor expression.

The habitat diversity of the fungus Nectria haematococca MPVI has been shown to be due in part to conditionally dispensable (CD) chromosomes that carry habitat-defining genes. From a biological perspective, the CD chromosomes are analogous to plasmids that possess genes that determine the habitats of plant-associated bacteria. This study establishes that the N. haematococca CD chromosome that contains the genes for Pea Pathogenicity (PEP cluster) also carries genes for the utilization of homoserine, an amino acid found in pea root exudates. Competition studies presented here demonstrate that an isolate that lacks the PEP cluster, but carries a portion of the CD chromosome containing the homoserine utilization (HUT) genes, is more competitive in the pea rhizosphere than an isolate without the CD chromosome. Further competition studies show that both the PDA1 and PDA6 CD chromosomes confer a competitive advantage in the rhizosphere of soybean, whereas only the PDA6 CD chromosome confers a competitive advantage in the rhizospheres of tomato and alfalfa, and only the PDA1 CD chromosome confers a competitive advantage in the rhizosphere of pea. These studies suggest the presence of genes on the PDA6 and PDA1 CD chromosomes that enhance the ability of N. haematococca to expand its habitat and support the idea that fungal CD chromosomes are analogous to host-specifying plasmids in plant-associated bacteria. Transformation, insertional mutagenesis, and bioinformatics were used to identify a cluster of five genes on the PDA1 CD chromosome that was responsible for the HUT phenotype in N. haematococca. One of the genes was found only in N. haematococca, another was a fungal transcription factor, and the other three had homologs involved in the synthesis of the amino acids methionine, threonine, and isoleucine, in which homoserine is an intermediate. Competition experiments that compared isolates with or without the HUT cluster showed that the HUT cluster is responsible for increased competitive ability of HUT+ N. haematococca isolates in the rhizosphere of pea. This study establishes that homoserine utilization can be a rhizosphere competency trait for N. haematococca and, to our knowledge, is the first example of a rhizosphere competency trait identified in a fungus.

Natural products occupy a central role as the majority of currently used antibiotic and anticancer agents. Among these are type-II polyketide synthase (PKS)-derived molecules, or polyketides, which are produced by many representatives of the genus Streptomyces. Some type-II polyketides, such as the tetracyclines and the anthracycline doxorubicin, are currently employed as therapeutics. However, several polyketide molecules exhibit promising biological activity, but due to toxic side effects or solubility concerns, remain undeveloped as drugs. Gilvocarcin V (GV) (topoisomerase II inhibitor) has a novel mechanism of action: [2+2] cycloaddition to thymine residues by the 8-vinyl side chain and cross-linking of histone H. Mithramycin blocks transcription of proto-oncogenes c-myc and c-src by forming an Mg2+-coordinated homodimer in the GC-rich minor groove of DNA. The purpose of this research was to investigate the biosynthesis of several type II polyketide compounds (e.g. chrysomycin, elloramycin, and mithramycin) with the goal of improving the bioactivities of these drugs through combinatorial biosynthesis. Alteration of the glycosylation pattern of these molecules is one promising way to improve or alter the bioactivities of these molecules. To this end, an understanding of the glycosyltransferases and post-polyketide tailoring enzymatic steps involved in these biosynthetic pathways must be established. Four specific aims were established to meet these goals. In specific aim 1, the biosynthetic locus of chrysomycin A was successfully cloned and elucidated, which afforded novel biosynthetic tools. Chrysomycin monooxygenases were found to catalyze identical roles to their gilvocarcin counterparts. Cloning of deoxysugar constructs (plasmids) which could direct biosynthesis of ketosugars, NDP-D-virenose, and NDP-D-fucofuranose in foreign pathways was undertaken in specific aim 2. Finally, these “sugar” plasmids were introduced into producer organisms of elloramycin and mithramycin pathways in specific aims 3 and 4 to interrogate the endogenous glycosyltransferases in order to alter their glycosylation patterns. These experiments resulted in the successful generation of a newly glycosylated tetracenomycin, as well as premithramycin, and mithramycin analogues. In specific aim 4, a new mithramycin analogue with an altered sugar pattern rationally designed and improved structural features was generated and structurally elucidated.

No description available.

Du fait de leurs propriétés immunostimulantes, les entérotoxines de Staphylococcus aureus (SEs) sont aussi considérées comme des outils thérapeutiques anticancéreux potentiels. Cependant, leurs implications dans de nombreuses pathologies humaines limitent leurs utilisations. Récemment, un opéron dénommé enterotoxin gene cluster (egc) codant pour cinq entérotoxines (SEG, SEI, SElM, SElN et SElO) supposées être de moindre virulence pour l’organisme, a été mis en évidence. En 2004, des patients atteints de carcinome broncho-pulmonaire ont été traités par l’administration d’un surnageant de culture d’une souche de S. aureus, contenant l’opéron egc. Ce traitement a permis d’allonger la durée de survie, et n’a eu aucun effet secondaire. Dans ce cadre, l’objectif de cette thèse a été d’étudier l’activité anti-tumorale des toxines de l’egc. Nos travaux ont mis en évidence l’activité tumoricide de ces toxines, induite par l’activation du système immunitaire. Cette toxicité est médiée par la sécrétion de nombreux médiateurs solubles comme le TNF-α et le NO. Nous avons confirmé le caractère pro-inflammatoire de type Th1 des toxines de l’egc. Nos travaux ont également montré qu’hormis SEI, les toxines de l’egc induisent des sécrétions de cytokines, chimiokines, protéases matricielles (MMPs) et facteurs de croissances nettement inférieures à celle induites par le reste des SEs. Ces résultats pourraient expliquer la faible toxicité associée aux toxines de l’egc. Enfin, nous avons montré que SElO possèdent une toxicité intrinsèque vis-à-vis des lignées tumorales. Cette étude plaide en faveur de l'intérêt des toxines de l’egc dans le développement de nouvelles approches en thérapie anti-tumorale / The use of classical superantigens (e.g. SEA, SEB and SEC) for treatment of cancer has resulted in a low response rates due to serious toxicity in humans. However, in a recent clinical study, remarkable results in treating lung cancer were obtained using superantigens encoded by the enterotoxin gene cluster (egc) without causing any significant toxicity. The current study was performed to investigate how egc superantigens (i.e. SEG, SEI, SElM, SElN and SElO) have tumoricidal activity with low toxicity. Indeed, we first demonstrated that tumoricidal activity of egc-SEs is mediated by immune cell activation, in particular, by secretion of soluble mediators such as nitric oxide and TNF-α. Thus, the proteomic analysis of the PBMC supernatants, showed that SEs-egc enhance the expression of pro-inflammatory cytokines, chimokines and many other biomarkers. Interestingly, levels were significantly higher in supernatants of SEA-stimulated PBMC than those with egc superantigens suggesting that staphylococcal superantigens differs in their inflammatory proprerties. Our results suggest that the relative lower pro-inflammatory activity of egc toxins may explain the low toxicity of these toxins observed during the clinical trial. Finally, we showed that SElO have a direct cytostatic activity against tumor cells. These findings suggest that egc-SEs seems to be good candidates for the development of new drugs in cancer therapy