Streptomyces coelicolor produces fatty acids for both primary metabolism and for production of the components of natural products such as undecylprodiginine. Primary metabolism makes the longer and predominantly branched-chain fatty acids, while undecylprodiginine utilizes shorter and almost exclusively straight chain fatty acids. The first step in fatty acid biosynthetic process is catalyzed by FabH (β-ketoacyl synthase III), which catalyzes a decarboxylative condensation of an acyl-CoA primer with malonyl-acyl carrier protein (ACP). The resulting 3-ketoacyl-ACP product is reduced by NADPH-dependent FabG into 3-hydroxyacyl-ACP, which is dehydrated by FabA to form enoyl-ACP. The NADH-dependent FabI (InhA) completes the cycle. Subsequent rounds of elongations in the pathways are catalyzed by the condensing enzyme FabF. For undecylprodiginine biosynthesis in S. coelicolor, homologues of the condensing enzymes (FabH and FabF) and the ACP (FabC) are encoded by redP, redR and redQ respectively in the red gene cluster. The genes encoding 3-ketoacyl-ACP reductase (FabG), 3-hydroxyacyl-ACP dehydratase (FabA), and enoyl-ACP reductase (FabI), are putatively shared between fatty acid and undecylprodigine biosynthesis, since the corresponding genes are not present within the red gene cluster of S. coelicolor. RedP is proposed to initiate biosynthesis of undecylprodiginine alkane chain by condensing an acetyl-CoA with a malonyl-RedQ, in contrast to FabH which process a broad range of acyl-CoA with malonyl-FabC. The 3-keto group of the resulting 3-ketoacyl-RedQ is then reduced to provide butyryl-RedQ, presumably by the type II FAS enzymes FabG, FabA and FabI. These enzymes would not differentiate between straight and branched-chain substrates, and have equal preference for FabC and RedQ ACPs. RedR would then catalyze four subsequent elongation steps with malonyl-RedQ, with appropriate 3-keto group processing after each step. The proposed role and substrate specificities of condensing enzymes RedP and FabH have not been investigated in S. coelicolor. The genes encoding FabG, FabA, and FabI have not been characterized in Streptomyces. Analysis of the S. coelicolor genome sequence has revealed the presence of one fabI gene (SCO1814, encoding an enoyl-ACP reductase), and three likely fabG genes (SCO1815, SCO1345, and SCO1346, encoding β-ketoacyl-ACP reductase). In the current study the substrates specificities of both RedP and FabH were determined from assays using pairings of two acyl-CoA substrates (acetyl-CoA and isobutyryl-CoA) and two malonyl-ACP substrates (malonyl-RedQ and malonyl-FabC) (FabC is a dedicated ACP for fatty acid biosynthesis and RedQ for undecylprodiginine biosynthesis in S. coelicolor). For RedP, activity was only observed with a pairing of acetyl-CoA and malonyl-RedQ. No activity was observed with isobutyryl-CoA consistent with the proposed role for RedP and the observation that acetyl CoA-derived prodiginines predominate in S. coelicolor. Malonyl-FabC is not a substrate for RedP, indicating that ACP specificity is one of the factors that permit a separation between prodiginine and fatty acid biosynthetic processes. In contrast to RedP, FabH was active with all pairings but demonstrated the greatest catalytic efficiency with isobutyryl-CoA using malonyl-FabC. Lower catalytic efficiency was observed using an acetyl-CoA and malonyl-FabC pairing consistent with the observation that in streptomycetes, a broad mixture of fatty acids are biosynthesized, with those derived from branched chain acyl-CoA starter units predominating. Diminished but demonstrable FabH activity was also observed using malonyl-RedQ, with the same preference for isobutyryl-CoA over acetyl-CoA, completing biochemical and genetic evidence that in the absence of RedP this enzyme can also play a role in prodiginine biosynthesis, producing branched alkyl chain prodiginines. The identification and characterization of both enzymes FabG and FabI was also carried out. A series of straight and branched-chain β-ketoacyl and enoyl substrates tethered to either NAC or ACP were synthesized and used to elucidate the functional role and substrate specificity of these enzymes. Kinetic analysis demonstrates that of the three S. coelicolor enzymes, SCO1815 and SCO1345 have NADPH-dependent β-ketoacyl-reductase activity, in contrast to SCO1346, which has NADH-dependent β-ketoacyl-reductase activity. Spectrophotometric assays revealed that all three FabGs are capable of utilizing both straight and branched-chain β-ketoacyl-NAC substrates. These results are consistent with FabGs role in fatty acid and undecylprodiginine biosynthesis, wherein it processes branched-chain for primary metabolism as well as straight-chain products for undecylprodiginine biosynthesis. LC/MS assays demonstrate that these FabG enzymes do not discriminate between primary metabolism ACP (FabC) and secondary metabolism ACP (RedQ) (except for SCO1345, which does not have any activity with RedQ). This relaxed substrate specificity allows these enzymes to process 3-ketoacyl-FabC substrates for fatty acid biosynthesis as well as 3-ketoacyl-RedQ substrates for undecylprodiginine biosynthesis. Similar to FabG, spectrophotometric and LC/MS assays were also carried out to elucidate the functional role and substrate specificity of S. coelicolor FabI. The kinetic analyses demonstrate that SCO1814 has NADH-dependent enoyl-ACP reductase activity. Spectrophotometric and LC/MS assays demonstrated that FabI does not differentiate between straight and branched-chain substrates, and has equal preference for FabC and RedQ ACPs. These observations provide experimental support for the hypothesis that these enzymes are shared and process the intermediates in the elongation cycle of both fatty acid and undecylprodiginine biosynthesis. In summary, these studies have demonstrated the activity of enzymes RedP, FabH, FabG and FabI (InhA) previously uncharacterized in S. coelicolor and clarified their role in fatty acid and undecylprodiginine biosynthesis.

No description available.

The effectiveness of the biocontrol product Actinovate® at enhancing tomato plant growth and yield, and reducing the presence of fungal pathogens was studied in greenhouse and field conditions. In the greenhouse, no differences were found among seed germination or plant survival rates, seedling heights, dry root weights, and dry shoot weights of tomato seedlings grown from seeds drenched with Actinovate® or Rootshield®. The effects of one initial Actinovate® seed drench at sowing, repeated applications through the drip irrigation throughout the season, or repeated applications through the drip irrigation plus foliar applications throughout the season at reducing plant infection by fungal plant pathogens, and increasing yield and quality for tomato plants (Solanum lycopersicum) were investigated in Los Alamos, CA, on a sandy loam soil. No significant differences in plant height were found among the four treatments. Marketable fruit weight was greater in the drip plus foliar treatment than in the Actinovate® seed drench treatment. The foliar plus drip treatment resulted in the greatest amount of powdery mildew present, although the disease pressure was low. No significant differences were found among the four treatments in the presence of Verticillium wilt or Sclerotinia.

No description available.

<p>Secondary metabolites produced by bacterial species serve many clinically useful purposes such as anti-bacterial, anti-cancer, and immunosuppresive agents. Actinobacteria, particularly the genus <em>Streptomyces</em>, have been an abundant source of such metabolites for the past half century. The production of secondary metabolites is controlled through vast regulatory cascades, but the activation and control of these pathways is still poorly understood. This leads to the inability to isolate all of the secondary metabolites that <em>Streptomyces</em> are capable of producing. This study focuses on the comparison of synthetic small molecules, which were found to alter the production of secondary metabolites in <em>S. coelicolor</em>. A comparative analysis of two of these molecules, ARC2 and ARC6, shows they modulate secondary metabolites in different ways. In a separate study, ARC2 was shown to achieve this phenotype through the inhibition of a target in fatty acid biosynthesis. The results of this study suggest that ARC6 does not have the same target, although it may target the same metabolic system. Furthermore, these two molecules also have opposite effects on <em>S. coelicolor </em>development. The cumulative results of this study suggest that ARC2 and ARC6 can act as separate chemical tools in enhancing the understanding of secondary metabolism.</p> / Master of Science (MSc)

<p>Secondary metabolites are vital to human health and strategies to improve their production and detection are equally essential. The blue pigmented metabolite actinorhodin produced by <em>Streptomyces coelicolor</em>, a genus renowned for their diverse secondary metabolites, provides a unique opportunity to identify small molecules probes of secondary metabolism. Small molecules capable of altering secondary metabolism will have widespread application in the streptomycetes due to their ease of addition to any culture condition. Taking advantage of the phenotypic versatility of the <em>S. coelicolor</em> lifecycle, we extended our search for small molecule modulators further to include the entire developmental process. In addition to alterations in secondary metabolism, these processes include growth inhibition, precocious sporulation and alterations in aerial hyphae formation and sporulation. This work provides the foundation for studying <em>Streptomyces</em> by chemical manipulation. Those compounds which stimulate secondary metabolism were narrowed down to 19 ARCs (for antibiotic remodeling compounds). From these, a set of 4 structurally related molecules, the ARC2 series, was identified as weak inhibitors of fatty acid biosynthesis and most likely lead to alterations in secondary metabolism through shifting precursors from primary to secondary metabolism. Consistent with the conservation of fatty acid biosynthesis within bacteria, the effect of the ARC2 series extends in general to the actinomycetes. This provides a simple strategy to alter the secondary metabolic profiles of a diverse range of actinomycetes.</p> / Doctor of Philosophy (PhD)

<p>The world is facing a public health threat due to increasing emergence of antibiotic resistance in pathogens. <em>Streptomyces </em>the soil-dwelling, Gram-positive, filamentous bacteria belonging to the family actinomycetes, are proven to be rich sources of natural antibiotics. Genome sequencing of <em>Streptomyces coelicolor, </em>a model organism of this genus, has revealed that in addition to the five antibiotics characterized so far, it possesses abundant genetic architecture of unexpressed biosynthetic or cryptic clusters for secondary metabolite production. The reason for their silence appears to be the poor understanding of their specific activation stimuli. In <em>Streptomyces coelicolor,</em> a pleiotropic regulator belonging to the two-component system family, <em>afsQ1</em>, has shown to activate the production of actinorhodin (ACT), undecylprodigiosin (RED), and calcium-dependent antibiotic (CDA). The aim of this research was to employ the genetically engineered <em>afsQ1</em> allele (named <em>afsQ1*</em>), which mimics the phosphorylated active form and obviates the need for specific external stimulus, and screen for novel antibiotic production. In this study, <em>afsQ1* </em>was introduced in various wild actinomycete isolates from the Wright Actinomycetes Collection (WAC) by conjugation and the resulting mutants were screened for antibiotic production. Two out of six WAC strains showed <em>afsQ1*- </em>induced antimicrobial activity. Interestingly, we were able to purify two antibiotic compounds, namely 1082 [M+2H]²⁺<strong> </strong>and 782 [M+H]⁺<strong> </strong>from the strain WAC00263. 1082 [M+2H]²⁺,<strong> </strong>a potentially novel antimicrobial peptide, exhibited activity against a wide range of Gram-positive bacteria including resistant pathogens such as vancomycin-resistant <em>Enterococcus</em> ATCC# 51299, a clinical isolate of methicillin resistant <em>Staphylococcus aureus</em>, and a clinical isolate of <em>S. aureus</em> BM3002. Moreover, it also showed activity against an opportunistic Gram-negative multi-drug resistant pathogen <em>Acinetobacter baumannii</em> B0098426R and a virulent strain of the fungus <em>Cryptococcus neoformans </em>H99<em>. </em>The second newly expressed molecule, 782 [M+H]⁺<strong> </strong>was not as potent as 1082 [M+2H]²⁺,<strong> </strong>so<strong> </strong>far only exhibited antimicrobial activity against the Gram-positive laboratory strains <em>Bacillus subtilis</em> #168 and <em>Micrococcus luteus</em>. These results reiterate that the technique of heterologous expression of the pleiotropic regulator, <em>afsQ1*</em>, in diverse actinomycetes is an excellent tool to induce novel antimicrobial production.</p> / Master of Science (MSc)

An increasing appreciation for the role of small RNA regulators prompted us to investigate the scope of RNA regulation in the bacterium, Streptomyces coelicolor. Our search revealed an antisense RNA that corresponds to the upstream region of four genes encoding cell wall cleavage enzymes (cell wall hydrolases), and a previously uncharacterized population of transfer RNA (tRNA) cleavage products. Further characterization of the 'tRNAs led to the discovery that S. coelicolor tRNAs are cleaved into 'tRNA halves' in a developmentally regulated fashion. All tRNAs seem to be susceptible to tRNA cleavage, although a bias was detected for tRNAs specifying highly used codons. To date, our work is the sole description of 'tRNA half production in a bacterium, and recent studies suggest that it is a widespread phenomenon among eukaryotic organisms. In a separate line of investigation, we noticed that a previous study had predicted that the genes associated with the antisense RNA are under the control of a riboswitch- a regulatory RNA element that directly controls gene expression in response to specific conditions. Our multifaceted characterization of this system began with the construction and phenotypic analyses of deletion mutant strains for several of the cell wall hydrolase-encoding genes. We demonstrate that S. coelicolor cell wall hydrolases are involved in germination, vegetative growth, and sporulation. Finally, we studied the potential for riboswitch regulation of one of the cell wall hydrolase-encoding genes, rpfA. RpfA is a resuscitation: Qromoting factor protein that is important for the revival of dormant bacteria, including the human pathogen and S. coelicolor relative - Mycobacterium tuberculosis. Our investigation uncovered evidence suggesting that the riboswitch region is involved in the regulation of rpfA, and we identified specific conditions under which it is repressed. This work represents a novel paradigm in the regulation of cell wall hydrolase expression. / Thesis / Doctor of Philosophy (PhD)

The growing problem of antibiotic resistance has prompted the need for new and novel antimicrobial therapies. The bacterial cell division pathway holds great promise for the development of novel broad-spectrum antibiotics as the majority of the proteins are essential for viability. The wealth of information regarding bacterial cell division has come from studies of the model organisms Escherichia coli and Bacillus subtilis. Although much has been elucidated regarding this pathway, the functions of many individual proteins remain unsolved. An important model organism for the investigation of cell division is Streptomyces coelicolor. The mycelial Streptomyces are sporulating, Gram-positive bacteria that grow in long branching networks of filamentous cells much like filamentous fungi. The normally essential process of cell division is dispensable for growth and viability of S. coelicolor. More interestingly, there are two different modes of cell division in this organism, one for vegetative growth and one is utilized for synchronous septation during sporulation. It is still unclear how developmental regulators control this switch, but advancements in fluorescence microscopy have shed some insight into the cell division process by allowing direct visualization of many cellular components and their dynamics. To better understand bacterial cell division and its regulation in S. coelicolor, three additional fluorescent proteins (FPs), including m.RFP, CyPet and YPet, have been established in this work. An m.RFP shuttle vector was constructed and the utility of m.RFP was tested by translationally fusing it to a tip-localizing protein, DiviVA. This work demonstrated that m.RFP is functional and an efficient marker for localized proteins. Also, established in this work is a two-colour fluorescence reporter system, which includes the fluorescent proteins CyPet and YPet that can be used to study co-localization and protein-protein interactions within cells. Future plans are to use co-localization of FP fusions and fluorescence resonance energy transfer (FRET) between CyPet and YPet to investigate the assembly of protein complexes within the cells, such as those involved in cell division. These studies will reveal critical information that is needed for the development of drugs that have novel mechanisms of action. / Thesis / Master of Science (MSc)

The Streptomyces are invaluable as a natural source of antibiotics and other bioactive compounds used in medicine and agriculture. S. coelicolor is the model streptomycete, and is studied for its complex secondary metabolism and multicellular life cycle. The subject of this work is bldB, a gene essential for development and antibiotic production in S. coelicolor, and one of its many homologues, located in the abaA antibiotic regulatory locus. The aim was to study the transcriptional regulation of bldB using a luminescent reporter, and investigate the role of each of the genes in the abaA cluster in regulation of antibiotic production, in order to understand the function and mechanism of action of bldB and its homologues. Individual deletion of each of the four genes in the abaA cluster resulted in varying effects on production of the antibiotic CDA. The bldB homologue, SCO0703, was shown to be a positive regulator of CDA, as the null mutant was severely defective in CDA production. It was found that bldB is expressed in most other bld developmental mutants, with the exception of bldD. There was no direct interaction observed between BldD and the bldB promoter, and possible mechanisms of indirect regulation are proposed. / Thesis / Master of Science (MSc)