Design and characterisation of synthetic operons for biohydrogen technology

Biohydrogen is produced by a number of microbial systems and the commonly used host bacterium Escherichia coli naturally produces hydrogen under fermentation conditions. One approach to engineering additional hydrogen production pathways is to introduce non-native hydrogenases into E. coli. An attractive candidate is the soluble [NiFe]-hydrogenase from Ralstonia eutropha, which has been shown to link NADH/NAD(+) biochemistry directly to hydrogen metabolism, an activity that E. coli does not perform. In this work, three synthetic operons were designed that code for the soluble hydrogenase and two different enzyme maturase systems. Interestingly, using this system, the recombinant soluble hydrogenase was found to be assembled by the native E. coli [NiFe]-hydrogenase assembly machinery, and, vice versa, the synthetic maturase operons were able to complement E. coli mutants defective in hydrogenase biosynthesis. The heterologously expressed soluble hydrogenase was found to be active and was shown to produce biohydrogen in vivo

Scarring of Florida’s seagrasses: assessment and management options

Management programs that address scarring of seagrasses should be based on an approach that involves (1) education, (2) channel marking, (3) increased enforcement, and (4) limited-motoring zones. Aerial monitoring and photography of the managed area are essential in evaluating the effectiveness of a program. Management programs that use this multifaceted approach have been instituted by a few local governments and at several state parks. Initial results of the programs indicate that in some areas seagrass scarring has been reduced but that in other areas emphasis may need to be increased on one or more of the components of the four-point approach. A statewide management plan is needed to address the most egregious scarring over large areas that may be difficult to regulate at the local-government level

Letter From Frank P. Sargent to Francis Mairs Huntington-Wilson, January 16th, 1908

In this letter to Francis Mairs Huntington Wilson, Frank P. Sargent, Commissioner General of Immigration, mentions a draft of a bill prepared in response to Wilson\u27s memorandum.https://digitalcommons.ursinus.edu/fmhw_third_documents/1021/thumbnail.jp

Tat signal peptide recognition during protein maturation and export

Nitrogen is one of the most abundant elements on Earth and mostly found in the atmosphere as the inert gas N2. Therefore the nitrogen cycle is important for maintaining the bioavailabilty of nitrogen for organisms. Denitrification is a process that closes the nitrogen cycle by subsequent conversion of nitrate to dinitrogen with reduction of nitrate to nitrite being the very first necessary step. The bacterial periplasmic nitrate reductase NapA is one of those nitrate reducing enzymes and contains a molybdenum cofactor and a [4Fe-4S] cluster as cofactors. As a periplasmic terminal reductase NapA has an N-terminal signal peptide harbouring a Tat (twin-arginine translocation) motif, which follows closely the consensus S/T-R-R-x-F-L-K. As with other proteins transported via the Tat pathway, NapA needs to be fully folded, and cofactor insertion needs to be completed, prior to export. This is assured by an individual chaperone in a process called ‘Tat-proofreading’. The proofreading chaperone for NapA is NapD, which had been previously shown to interact tightly with the signal peptide of NapA.In this work the binding epitope on the Escherichia coli NapA signal peptide recognised by NapD was mapped for the first time. The key amino acid residues (NapA R6, K10, A17) overlapped with the Tat targeting motif and were further characterized in vitro and in vivo for their importance in NapD binding, Tat transport and NapA biosynthesis. In addition, napD suppressor mutants able to re-bind the NapA A17Q variant were isolated. NMR spectroscopy revealed the 3D solution structure of NapD in complex with the NapA signal peptide. Interestingly, the signal peptide of NapA is a-helical when bound to NapD. Overall, the structure supports strongly that NapA residues R6, K10 and A17 interact with NapD. Pulsed EPR spectroscopy on the isolated signal peptide indicated structural changes of the NapA signal peptide between NapD bound and unbound states, though it was believed that an overall a-helical structure was maintained. Co-purification studies of the complete NapDA complex for crystallisation trials resulted in increased information on the behaviour of the complex and the order of cofactor insertion into NapA. Finally, an in vitro translation and cross-linking approach was attempted with the aim of addressing whether direct contact was made between NapD and the Tat translocase. In addition, functional chromosomal fusions of either NapD or NapA with fluorescent proteins were generated to form a basis for a future project based on fluorescence correlation spectroscopy in living cells. This project has therefore provided fresh insight into the NapA-NapD interaction at the molecular level and laid the foundations for future research in this area.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Assigning output variables to equations using linear programming

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98133/1/690200231_ftp.pd

Biosynthesis of selenate reductase in <i>Salmonella enterica</i>:critical roles for the signal peptide and DmsD

Salmonella enterica serovar Typhimurium is a Gram-negative bacterium with a flexible respiratory capability. Under anaerobic conditions, S. enterica can utilize a range of terminal electron acceptors, including selenate, to sustain respiratory electron transport. The S. enterica selenate reductase is a membrane-bound enzyme encoded by the ynfEFGH-dmsD operon. The active enzyme is predicted to comprise at least three subunits where YnfE is a molybdenum-containing catalytic subunit. The YnfE protein is synthesized with an N-terminal twin-arginine signal peptide and biosynthesis of the enzyme is coordinated by a signal peptide binding chaperone called DmsD. In this work, the interaction between S. enterica DmsD and the YnfE signal peptide has been studied by chemical crosslinking. These experiments were complemented by genetic approaches, which identified the DmsD binding epitope within the YnfE signal peptide. YnfE signal peptide residues L24 and A28 were shown to be important for assembly of an active selenate reductase. Conversely, a random genetic screen identified the DmsD V16 residue as being important for signal peptide recognition and selenate reductase assembly

Expanding the substrates for a bacterial hydrogenlyase reaction

Escherichia coli produces enzymes dedicated to hydrogen metabolism under anaerobic conditions. In particular, a formate hydrogenlyase (FHL) enzyme is responsible for the majority of hydrogen gas produced under fermentative conditions. FHL comprises a formate dehydrogenase (encoded by fdhF) linked directly to [NiFe]-hydrogenase-3 (Hyd-3), and formate is the only natural substrate known for proton reduction by this hydrogenase. In this work, the possibility of engineering an alternative electron donor for hydrogen production has been explored. Rational design and genetic engineering led to the construction of a fusion between Thermotoga maritima ferredoxin (Fd) and Hyd-3. The Fd-Hyd-3 fusion was found to evolve hydrogen when co-produced with T. maritima pyruvate :: ferredoxin oxidoreductase (PFOR), which links pyruvate oxidation to the reduction of ferredoxin. Analysis of the key organic acids produced during fermentation suggested that the PFOR/Fd-Hyd-3 fusion system successfully diverted pyruvate onto a new pathway towards hydrogen production

Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone

Escherichia coliis a Gram-negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the periplasmic nitrate reductase, NapA, contains two types of redox cofactor and is exported across the cytoplasmic membrane by the twin-arginine protein transport pathway. NapD is a small cytoplasmic protein that is essential for the activity of the periplasmic nitrate reductase and binds tightly to the twin-arginine signal peptide of NapA. Here we show, using spin labelling and EPR, that the isolated twin-arginine signal peptide of NapA is structured in its unbound form and undergoes a small but significant conformational change upon interaction with NapD.In addition, a complex comprising the full-length NapA protein and NapD could be isolated by engineering an affinity tag onto NapD only. Analytical ultracentrifugation demonstrated that the two proteins in the NapDA complex were present in a 1:1 molar ratio, and small angle X-ray scattering analysis of the complex indicated that NapAwas at least partially folded when bound by its NapD partner. A NapDA complex could not be isolated in the absence of the NapA Tat signal peptide. Taken altogether, this work indicates that the NapD chaperone binds primarily at the NapA signal peptide in this system and points towards a role for NapD in the insertion of the molybdenum cofactor