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Genetic and Biochemical Investigation of the Siderophore Biosynthesis Pathway in Haloferax volcanii

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Title:
Genetic and Biochemical Investigation of the Siderophore Biosynthesis Pathway in Haloferax volcanii
Series Title:
18th Annual Undergraduate Research Symposium
Creator:
Al-Rubaee, Nadia
Language:
English

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Center for Undergraduate Research
Biological Sciences
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Conference papers and proceedings
poster ( aat )

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Abstract:
Iron plays a crucial role in cellular pathways as a metabolic cofactor and electron carrier in ATP synthesis. Under low iron conditions, many microorganisms have the ability to synthesize and secrete measurable quantities of siderophores. These low molecular weight chelators form complexes with free iron, which can then be taken up by the organism. In bacteria and eukaryotes, the regulation mechanisms associated with iron homeostasis have been studied extensively and are clearly distinguished. In archaea, however, the products and pathways associated with siderophore biosynthesis are not well characterized. In a model archaeon, Haloferax volcanii, two gene homologs associated with the Escherichia coli aerobactin biosynthesis pathway were proposed to function in an iron uptake component (IUC) system. Comparative genomic and genetic analyses were employed to target the genes for deletion, followed by trans-complementation to reconstruct the pathway. Chrome azurol S assays were used as part of a biochemical approach to evaluate siderophore production in the wild type and deletion strains under low iron conditions. The results indicated that the targeted iuc gene homologs of iucA and iucC were key in siderophore production. These findings provide a novel understanding of archaeal homeostasis mechanisms and the biological pathways involved in extracellular iron acquisition. ( en )
General Note:
Research Authors: Nadia Al-Rubaee, Sungmin Hwang, Julie A. Maupin-Furlow - University of Florida ; Paula Mondragon - Miami Dade College
General Note:
Faculty Mentor: Julie Maupin-Furlow - Microbiology and Cell Science, University of Florida

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University of Florida
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Copyright Nadia Al-Rubaee. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.

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Genetic and Biochemical Investigation of the Siderophore Biosynthesis Pathway in Haloferax volcanii Nadia Al Rubaee, 1 Sungmin Hwang, 1 Paula Mondragon, 2 Julie Maupin Furlow 1 1 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 2 Department of Biology, Miami Dade College, Miami, FL Methodology Conclusions References Introduction Results Acknowledgements Iron is a key component in biological systems Because iron can exist in several oxidation states, it has the ability to act as a redox center for metalloproteins and electron carriers in the electron transport chain As an ion, iron can also function as a cofactor Iron sulfur clusters are used by aconitase in the citric acid cycle, showing the multifaceted role of iron in ATP synthesis When cellular iron levels are exceedingly high, iron can be toxic ; thus, many microorganisms have regulatory mechanisms to combat this toxicity Alternatively, low iron conditions can also be dangerous because iron cannot be synthesized in vivo When environmental iron levels are low, many microorganisms employ an iron uptake component (IUC) system and synthesize iron chelators termed siderophores that are low molecular weight but high affinity Once secreted into the environment, siderophores bind and sequester iron This complex can then be taken up by the microorganism for use In bacteria and eukaryotes, the regulatory mechanisms associated with iron homeostasis have been studied at length and show clear distinctions With regards to Archaea the third domain of life, the siderophore biosynthesis pathway is poorly understood Through the results of the CAS assay, overlay CAS assay, and liquid CAS assay, it is evident that the H 26 strain of Haloferax volcanii produces siderophores These assays also showed that the deletion of the hvo_B 0041 gene resulted in lack of siderophore production, indicating an impairment in the siderophore biosynthesis pathway Deletion of the hvo_B 0044 gene yielded the same results After the complementation of the hvo_B 0041 gene, CAS assays indicated siderophore production This result implies restoration of pathway function and confirms that there were no polar effects following the deletion of the gene After complementation of the hvo_B 004 4 gene in the deletion strain, the liquid CAS assay did not confirm siderophore production Polar effects could not be ruled out Overall, there is sufficient evidence to propose the involvement of both the hvo_B 0041 and hvo_B 0044 genes in the siderophore biosynthesis pathway Implementing a markerless in frame deletion based on the pyrE 2 pop in/pop out method hvo_B 0044 and hv 0 _B 0041 were targeted to generate the gene deletion strains from the parent strain H 26 The strains were plated on Hv Ca medium, and a bl ue dye was poured as an overlay to simulate traditional CAS plating without the need for isolation of the supernatant Plates were incubated and subsequently examined for a yellow color change Chrome azurol S (CAS) assays were used to detect siderophore production S trains were grown in Hv Ca liquid medium Cell growth was monitored by OD 600 nm Stationary phase cells were harvested by centrifugation Culture broth ( 500 l of supernatant) was transferred to a new tube Aliquots were spotted onto 3 mm filter paper discs The plates were incubated at 42 o C Results of the CAS assay can be seen in Figure 3 Complimentary strains were generated as follows Complementation plasmids pJAM 3176 and pJAM 3177 carrying hvo_boo 41 and hvo_b 0044 genes, respectively, were constructed and transformed into the appropriate deletion strains Transformants were selected on ATCC 974 medium containing novobiocin Approximately 10 colonies were isolated and replated from each transformation ( hvo_B 0041 /pJAM 3176 and hvo_B 0044 /pJAM 3177 ) Cells carrying the plasmids were screened by PCR and gel electrophoresis The Hfx volcanii parent, mutant and complemented strains listed in Table 1 were grown in 4 mL of PR media ( iron, BPS) at 42 o C Cells were harvested at OD 600 nm of 0 5 to 0 7 by centrifugation The c ulture broth ( 500 l of supernatant) was transferred to a new tube A volume of 200 l of supernatant was mixed with 200 l of 1 x CAS liquid and incubated Results were observed and can be seen in Figure 4 1. Allers T., Ngo, H., Mevarech M., & Lloyd, R. G. (2004). Development of Additional Selectable Markers for the Halophilic Archaeon Haloferax volcanii Based on the leuB and trpA Genes. Applied and Environmental Microbiology,70 (2), 943 953. 2. Miranda, H. V., Nembhard N., Su, D., & Maupin Furlow J. A. (2011). E1 and ubiquitin like proteins provide a direct link between protein conjugation and sulfur transfer in archaea. Proc Natl Acad Sci U S A 108: 4417 4422. Hypothesis In a model archaeon, Haloferax volcanii gene homologs associated with the Escherichia coli aerobactin biosynthesis pathway were proposed to function in an iron uptake component system Investigating the H 26 strain of Haloferax volcanii, the hvo_B 0044 and hv 0 _B 0041 genes are homologs of iucA and iucC respectively These gene homologs were the focus of this study Future Work The regulation mechanisms of the two iucA and iucC homologs have not been precisely determined and future research into these processes would provide clarity in a broader viewpoint Additionally, a point of interest is the chemical composition of the Haloferax volcanii siderophore For future study, it is recommended that the siderophore product from the CAS assay be isolated for the purposes of identification Purification of the siderophore by liquid chromatography followed by NMR and mass spectrometry (MS) analyses are approaches that could be implemented with this goal in mind Table 1. Strains used in this study Figure 3: CAS plate assay for siderophore detection. Yellow halo was observed after incubation at 42 o C for 89 h (left) and 121 h (right). Ctrl (medium) H26 iucC iucA iucAC ubaA samp 1/2/3 ubaA samp 1/2/3 entB iucC + IucC iucA + IucA dat bdb iucABDC iucABDC dat bdb Figure 4: CAS liquid assay for siderophore detection. Color change was recorded after incubation at 42C for 18 h. PCR analysis of the hvo_B 0041 /pJAM 3176 and hvo_B 0044 /pJAM 3177 transformants ( 4 replications each) revealed DNA fragments ~ 2 kbp in length This result is consistent with the length of the complemented genes and indicates that the transformation was successful ( hvo_B 0041 is 1 839 nt long, hvo_B 0044 is 1 782 nt long) The overlay CAS and CAS plate assays showed yellow rings (halos) surrounding the H 26 colonies No halos were associated with the hvo_B 0041 and hvo_B 0044 mutants in either assay Furthermore, the Hv Ca medium showed no halo in the CAS plate assay, ensuring a negative control In terms of the liquid assay, the blue dye became pink in the H 26 strain and the following mutants : ubaA samp 1 / 2 / 3 ubaA samp 1 / 2 / 3 entB hvo_B 0041 + HVO_B 0041 (complemented strain) Figure 1: Hal oferax volcanii gene cluster proposed to be involved in siderophore biosynthesis. GI accession number, gene locus tag, and base pair scale are shown Figure 2: Strategy for pyrE2 gene deletion in Hal oferax volcanii. 1 abc hvo_b0047 dat b0046 bdb b0045 iucA b0044 iucB b0043 iucD b0042 iucC b0041 arcR14 b0040 2000 bp Strain Description Reference H26 DS70 pyrE2 [1] iucC H26 hvo_b0041 This study iucA H26 hvo_b0044 This study iucAC H26 hvo_b0041 hvo_b0044 This study ubaA H26 hvo_b0044 [2] samp1/2/3 H26 hvo_2619 hvo_0202 hvo_2177 [2] ubaA samp1/2/3 H26 hvo_0558 hvo_2619 hvo_0202 hvo_2177 [2] entB H26 hvo_ 2328 Maupin lab iucC +IucC iucC containing in trans HVO_B0041 expression This study iucA +IucA iucA containing in trans HVO_B0044 expression This study dat bdb iucABDC H26 hvo_b0046 41 This study iucABDC H26 hvo_b0044 41 This study dat bdb H26 hvo_b0046 45 This study This work was funded in part by the National Institutes of Health (R 01 GM 57498 ) and the Department of Energy (DE FG 02 05 ER 15650 ) to J M F P M was awarded by the National Science STEM Talent Expansion program Discussion