Ahmet Bozdag¹
Coauthor: Michael C. Flickinger¹,²,³
Graduate Programs: Microbiology¹; Chemical & Biomolecular Engineering²; Golden LEAF Biomanufacturing & Training Education Center, NCSU³
Advisor: Michael C. Flickinger
Poster Number: 16
Title: Formaldehyde Tolerance and Carbon Dissimilation by Thermotolerant Methylotrophic Strains of Bacillus methanolicus strains
MGA3 and PB1
Abstract
Bacillus methanolicus is a gram-positive aerobic methylotroph growing optimally at 50-53 °C. Wild-type strains of B. methanolicus have been reported to secrete 58 g/l of L-glutamate in fed-batch cultures while classical mutants can secrete 37 g/l of L-lysine, at 50°C. Methylotrophy in B. methanolicus is encoded by an endogenous plasmid, pBM19 in strain MGA3, except for hexulose phosphate synthase (hps) and phosphohexuloisomerase (phi). It is a promising candidate for industrial production of chemical intermediates or amino acids from methanol (MeOH).
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B. methanolicus employs the ribulose monophosphate (RuMP) pathway to assimilate the carbon derived from MeOH, but enzymes that dissimilate carbon have not been identified. Formaldehyde and formate were identified as intermediates of MeOH dissimilation by ¹³C NMR. This study aims to elucidate and compare the carbon dissimilation pathways of
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B. methanolicus strains MGA3 and PB1. Formaldehyde tolerance of B. methanolicus strains MGA3 and PB1 were determined by growth inhibition studies using formaldehyde with 0.5, 1 and 2 mM final concentrations. Fedbatch fermentations of B. methanolicus strains MGA3 and PB1 were conducted in 2-liter fermentation vessels and dissimilated MeOH carbon was detected in exhaust gas by CO2 sensor. The proportion of dissimilated MeOH carbon was calculated by mass balance. Southern blot analysis was used to show the presence of formaldehyde (faldh) and formate dehydrogenase (fdh) genes by using B. licheniformis, B. kaustophilus and B. subtilis faldh and fdh genes as probes. Growth inhibition studies demonstrate that B. methanolicus PB1 can tolerate higher concentration of formaldehyde compared to MGA3. MeOH fed-batch fermentations show that ~40% of the methanol carbon is dissimilated as carbon dioxide and that strain PB1 dissimilates more carbon than MGA3. Southern blot experiments demonstrated the presence of the faldh and fdh in strain PB1 but not in MGA3. These results and genome analysis suggest that B. methanolicus strains MGA3 and PB1 are different in terms of formaldehyde metabolism and favor strain MGA3 over PB1 as a candidate for production of intermediates and amino acids from MeOH.
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Samanthi S. Kottegoda
Coauthor: Michael R.Hyman
Graduate Program: Microbiology
Advisor: Michael R. Hyman
Poster Number: 94
Title: Metabolism of Isobutane by Mycobacterium austroafricanum JOB5
Abstract
Isobutane, the simplest branched alkane, is an important component of gasoline, liquefied petroleum gas and natural gas. Few isobutane-oxidizing organisms have been identified and little is known about either the pathway or the enzyme responsible for initiating isobutane oxidation. The only previous study of the aerobic microbial isobutane oxidation suggested that propane and isobutane are oxidized by two different oxygenase enzymes. In this study, we have compared oxidation of propane and isobutane by Mycobacterium austroafricanum JOB5. The initial steps of oxygenase-dependent alkane-oxidations, as well as the further metabolites generated from the alcohol products of these reactions were compared at the physiological level. The isobutane-dependent induction of isobutane-oxidizing activity in fructose-grown cells was also examined using tert-butyl alcohol production as an indicator. Finally, changes in the proteins produced by whole cells during induction of isobutaneoxidizing activity, as well as the proteins produced by cells grown on propane, isobutane and their respective primary alcohol products were examined.
Cells grown on either propane or isobutane were able to oxidize both of these alkanes at high rates without any delay and were fully and consistently inhibited by acetylene. Oxidation of primary alcohol products of these reactions by both cell types was also very similar. De novo synthesis of a single 53kDa polypeptide was observed during the induction of isobutane-oxidizing activity
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in fructose grown cells. This polypeptide and another major 38kDa polypeptide were present at varying levels in cells grown on propane, isobutane, 1-propanol or 2-methyl-1-propanol and neither polypeptide was present in fructose-grown cells. Based on these results we conclude that propane- and isobutane-grown cells are very similar both in terms of their catalytic capabilities and whole cell protein expression. We further conclude that both 53 and 38kDa polypeptides are likely to be important structural components of the oxygenase enzyme responsible for initiating both propane and isobutane oxidation.
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Akinbolade O. Oyegunwa
Coauthors: Michael L. Sikes, Jason Ray Wilson, Frank Scholle, and Scott M. Laster
Graduate Program: Microbiology
Advisor: Scott M. Laster
Poster Number: 130
Title: Tetra-O-methyl nordihydroguaiaretic acid (Terameprocol) inhibits the NF-κB-dependent transcription of TNF-α and MCP-1/CCL2 genes by preventing RelA from binding its cognate sites on DNA
Abstract
Tetra-O-methyl nordihydroguaiaretic acid also known as Terameprocol (TMP), is a naturally occurring phenolic compound found in the resin of the creosote bush.
We have shown previously that TMP will suppress production of certain inflammatory cytokines, chemokines and lipids from macrophages
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following stimulation with LPS or infection with H1N1 influenza virus. In this study our goal was to elucidate the mechanism underlying TMP-mediated suppression of cytokine and chemokine production. We focused our investigations on the response to LPS and the NF-κB protein RelA a transcription factor whose activity is critical to LPS responsiveness. (continued in next column >>)
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Reporter assays were performed with HEK293 cells overexpressing either TLR-3, -4, or -8 and a plasmid containing the luciferase gene under control of an |
NF-κB response element. Cells were then treated with LPS, poly(I:C), or resiquimod, and/or TMP, and lysates were measured for luciferase activity. ChIP assays using RelA specific antibodies showed TMP caused virtually complete inhibition of RelA binding in vivo to promoters for the genes TNF-α, MCP-1/CCL2, and RANTES/CCL5 although the LPS-dependent synthesis of IκB-α was not inhibited. EMSA assays did not reveal an effect of TMP on the binding of RelA to naked DNA templates in vitro. Furthermore, TMP did not inhibit the nuclear translocation of NF-κB RelA nor the phosphorylation of IκB-α. Our results strongly suggest that TMP acts indirectly as an inhibitor of NF-κB-dependent transcription by preventing RelA from binding the promoters of certain key cytokine and chemokine genes. |
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