HYDROGEN AS FUEL FOR ENTERIC BACTERIA: BIOCHEMISTRY OF THE MEMBRANE BOUND NIFE HYDROGENASE

Hydrogen is considered both a fuel for the future and an ancient fuel for life. Hydrogenases catalyse the interconversion of molecular hydrogen, H2, and protons, H+. A subgroup of hydrogenases, the membrane bound NiFe hydrogenases (MBH), have been the subject of much research interest. This is firstly due to their possible applications in biotechnology, but also because they have been implicated in the virulence of gut pathogens. The MBH are divided into O2 tolerant and O2 sensitive based on their ability to catalyse H2 oxidation in the presence of O2, and the two classes are both structurally and mechanistically distinct. Understanding these distinctions is important both for technology which aims to achieve more minimal models of enzymes, but also for relating the way different MBH are expressed at different stages of infection. The properties of variants of two O2 tolerant MBH, Escherichia coli Hyd 1 and Salmonella enterica Hyd 5 and one O2 sensitive MBH, E. coli Hyd 2, have been examined with regards to how the properties of specific amino acids achieve control over catalysis and O2 tolerance. Red®/ET® recombination methodology has been applied for the first time to hydrogenases. This methodology allows the rapid generation of hydrogenase knockouts and single site variants in E. coli in addition to the incorporation of polyhistidine tags to enable protein purification. Purified native and variant hydrogenases have been studied with protein film electrochemistry. Hyd 1 and Hyd-5 E73A and H229A variants were shown to have diminished O2 tolerance whilst a Hyd 1 E73Q variant had an increased catalytic bias towards H2 production. It was established that Hyd 1 was expressed during growth in glucose limited minimal media, although no change in growth rate or competitive ability was seen in a Hyd 1 knockout strain

The antimicrobial activity of a carbon monoxide releasing molecule (EBOR-CORM-1) is shaped by intraspecific variation within3 Pseudomonas aeruginosa populations

Carbon monoxide releasing molecules (CORMs) have been suggested as a new synthetic class of antimicrobials to treat bacterial infections. Here we utilized a novel EBOR-CORM-1 ([NEt4][MnBr2(CO)4]) capable of water-triggered CO-release, and tested its efficacy against a collection of clinical Pseudomonas aeruginosa strains that differ in infection-related virulence traits. We found that while EBOR-CORM-1 was effective in clearing planktonic and biofilm cells of P. aeruginosa strain PAO1 in a concentration dependent manner, this effect was less clear and varied considerably between different P. aeruginosa cystic fibrosis (CF) lung isolates. While a reduction in cell growth was observed after 8 h of CORM application, either no effect or even a slight increase in cell densities and the amount of biofilm was observed after 24 h. This variation could be partly explained by differences in bacterial virulence traits: while CF isolates showed attenuated in vivo virulence and growth compared to strain PAO1, they formed much more biofilm, which could have potentially protected them from the CORM. Even though no clear therapeutic benefits against a subset of isolates was observed in an in vivo wax moth acute infection model, EBOR-CORM-1 was more efficient at reducing the growth of CF isolate co-culture populations harboring intraspecific variation, in comparison with efficacy against more uniform single isolate culture populations. Together these results suggest that CORMs could be effective at controlling genetically diverse P. aeruginosa populations typical for natural chronic CF infections and that the potential benefits of some antibiotics might not be observed if tested only against clonal bacterial populations

Parallel specification of competing sensorimotor control policies for alternative action options.

Recent theory proposes that the brain, when confronted with several action possibilities, prepares multiple competing movements before deciding among them. Psychophysical supporting evidence for this idea comes from the observation that when reaching towards multiple potential targets, the initial movement is directed towards the average location of the targets, consistent with multiple prepared reaches being executed simultaneously. However, reach planning involves far more than specifying movement direction; it requires the specification of a sensorimotor control policy that sets feedback gains shaping how the motor system responds to errors induced by noise or external perturbations. Here we found that, when a subject is reaching towards multiple potential targets, the feedback gain corresponds to an average of the gains specified when reaching to each target presented alone. Our findings provide evidence that the brain, when presented with multiple action options, computes multiple competing sensorimotor control policies in parallel before implementing one of them

Retuning the Catalytic Bias and Overpotential of a [NiFe]-Hydrogenase via a Single Amino Acid Exchange at the Electron Entry/Exit Site

The redox chemistry of the electron entry/exit site in Escherichia coli hydrogenase-1 is shown to play a vital role in tuning biocatalysis. Inspired by nature, we generate a HyaA-R193L variant to disrupt a proposed Arg-His cation-π interaction in the secondary coordination sphere of the outermost, "distal", iron-sulfur cluster. This rewires the enzyme, enhancing the relative rate of H 2 production and the thermodynamic efficiency of H 2 oxidation catalysis. On the basis of Fourier transformed alternating current voltammetry measurements, we relate these changes in catalysis to a shift in the distal [Fe 4S 4] 2+/1+ redox potential, a previously experimentally inaccessible parameter. Thus, metalloenzyme chemistry is shown to be tuned by the second coordination sphere of an electron transfer site distant from the catalytic center

How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase

Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His(229) from the large subunit was close to the proximal [4Fe–3S] cluster in the small subunit. In addition, His(229) was observed to lie close to a buried glutamic acid (Glu(73)), which is conserved in oxygen-tolerant hydrogenases. His(229) and Glu(73) of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His(229) or Glu(73) with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His(229) has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu(73)could play a supporting role in fine-tuning the chemistry of His(229) to enable this function

Re-engineering a NiFe hydrogenase to increase the H2 production bias while maintaining native levels of O2 tolerance

Naturally occurring oxygen tolerant NiFe membrane bound hydrogenases have a conserved catalytic bias towards hydrogen oxidation which limits their technological value. We present an Escherichia coli Hyd-1 amino acid exchange that apparently causes the catalytic rate of H2 production to double but does not impact the O2 tolerance

A Tale of Three Species: Adaptation of Sodalis glossinidius to Tsetse Biology, Wigglesworthia Metabolism, and Host Diet.

The tsetse fly is the insect vector for the Trypanosoma brucei parasite, the causative agent of human African trypanosomiasis. The colonization and spread of the trypanosome correlate positively with the presence of a secondary symbiotic bacterium, Sodalis glossinidius The metabolic requirements and interactions of the bacterium with its host are poorly understood, and herein we describe a metabolic model of S. glossinidius metabolism. The model enabled the design and experimental verification of a defined medium that supports S. glossinidius growth ex vivo This has been used subsequently to analyze in vitro aspects of S. glossinidius metabolism, revealing multiple unique adaptations of the symbiont to its environment. Continued dependence on a sugar, and the importance of the chitin monomer N-acetyl-d-glucosamine as a carbon and energy source, suggests adaptation to host-derived molecules. Adaptation to the amino acid-rich blood diet is revealed by a strong dependence on l-glutamate as a source of carbon and nitrogen and by the ability to rescue a predicted l-arginine auxotrophy. Finally, the selective loss of thiamine biosynthesis, a vitamin provided to the host by the primary symbiont Wigglesworthia glossinidia, reveals an intersymbiont dependence. The reductive evolution of S. glossinidius to exploit environmentally derived metabolites has resulted in multiple weaknesses in the metabolic network. These weaknesses may become targets for reagents that inhibit S. glossinidius growth and aid the reduction of trypanosomal transmission.IMPORTANCE Human African trypanosomiasis is caused by the Trypanosoma brucei parasite. The tsetse fly vector is of interest for its potential to prevent disease spread, as it is essential for T. brucei life cycle progression and transmission. The tsetse's mutualistic endosymbiont Sodalis glossinidius has a link to trypanosome establishment, providing a disease control target. Here, we describe a new, experimentally verified model of S. glossinidius metabolism. This model has enabled the development of a defined growth medium that was used successfully to test aspects of S. glossinidius metabolism. We present S. glossinidius as uniquely adapted to life in the tsetse, through its reliance on the blood diet and host-derived sugars. Additionally, S. glossinidius has adapted to the tsetse's obligate symbiont Wigglesworthia glossinidia by scavenging a vitamin it produces for the insect. This work highlights the use of metabolic modeling to design defined growth media for symbiotic bacteria and may provide novel inhibitory targets to block trypanosome transmission

A Tale of Three Species: Adaptation of Sodalis glossinidius to Tsetse Biology, Wigglesworthia Metabolism, and Host Diet.

The tsetse fly is the insect vector for the Trypanosoma brucei parasite, the causative agent of human African trypanosomiasis. The colonization and spread of the trypanosome correlate positively with the presence of a secondary symbiotic bacterium, Sodalis glossinidius The metabolic requirements and interactions of the bacterium with its host are poorly understood, and herein we describe a metabolic model of S. glossinidius metabolism. The model enabled the design and experimental verification of a defined medium that supports S. glossinidius growth ex vivo This has been used subsequently to analyze in vitro aspects of S. glossinidius metabolism, revealing multiple unique adaptations of the symbiont to its environment. Continued dependence on a sugar, and the importance of the chitin monomer N-acetyl-d-glucosamine as a carbon and energy source, suggests adaptation to host-derived molecules. Adaptation to the amino acid-rich blood diet is revealed by a strong dependence on l-glutamate as a source of carbon and nitrogen and by the ability to rescue a predicted l-arginine auxotrophy. Finally, the selective loss of thiamine biosynthesis, a vitamin provided to the host by the primary symbiont Wigglesworthia glossinidia, reveals an intersymbiont dependence. The reductive evolution of S. glossinidius to exploit environmentally derived metabolites has resulted in multiple weaknesses in the metabolic network. These weaknesses may become targets for reagents that inhibit S. glossinidius growth and aid the reduction of trypanosomal transmission.IMPORTANCE Human African trypanosomiasis is caused by the Trypanosoma brucei parasite. The tsetse fly vector is of interest for its potential to prevent disease spread, as it is essential for T. brucei life cycle progression and transmission. The tsetse's mutualistic endosymbiont Sodalis glossinidius has a link to trypanosome establishment, providing a disease control target. Here, we describe a new, experimentally verified model of S. glossinidius metabolism. This model has enabled the development of a defined growth medium that was used successfully to test aspects of S. glossinidius metabolism. We present S. glossinidius as uniquely adapted to life in the tsetse, through its reliance on the blood diet and host-derived sugars. Additionally, S. glossinidius has adapted to the tsetse's obligate symbiont Wigglesworthia glossinidia by scavenging a vitamin it produces for the insect. This work highlights the use of metabolic modeling to design defined growth media for symbiotic bacteria and may provide novel inhibitory targets to block trypanosome transmission

NASA SpaceCube Next-Generation Artificial-Intelligence Computing for STP-H9-SCENIC on ISS

Recently, Artificial Intelligence (AI) and Machine Learning (ML) capabilities have seen an exponential increase in interest from academia and industry that can be a disruptive, transformative development for future missions. Specifically, AI/ML concepts for edge computing can be integrated into future missions for autonomous operation, constellation missions, and onboard data analysis. However, using commercial AI software frameworks onboard spacecraft is challenging because traditional radiation-hardened processors and common spacecraft processors cannot provide the necessary onboard processing capability to effectively deploy complex AI models. Advantageously, embedded AI microchips being developed for the mobile market demonstrate remarkable capability and follow similar size, weight, and power constraints that could be imposed on a space-based system. Unfortunately, many of these devices have not been qualified for use in space. Therefore, Space Test Program - Houston 9 - SpaceCube Edge-Node Intelligent Collaboration (STP-H9-SCENIC) will demonstrate inflight, cutting-edge AI applications on multiple space-based devices for next-generation onboard intelligence. SCENIC will characterize several embedded AI devices in a relevant space environment and will provide NASA and DoD with flight heritage data and lessons learned for developers seeking to enable AI/ML on future missions. Finally, SCENIC also includes new CubeSat form-factor GPS and SDR cards for guidance and navigation

Biosynthesis of Salmonella enterica [NiFe]-hydrogenase-5 : probing the roles of system-specific accessory proteins

A subset of bacterial [NiFe]-hydrogenases have been shown to be capable of activating dihydrogen-catalysis under aerobic conditions; however, it remains relatively unclear how the assembly and activation of these enzymes is carried out in the presence of air. Acquiring this knowledge is important if a generic method for achieving production of O2-resistant [NiFe]-hydrogenases within heterologous hosts is to be developed. Salmonella enterica serovar Typhimurium synthesizes the [NiFe]-hydrogenase-5 (Hyd-5) enzyme under aerobic conditions. As well as structural genes, the Hyd-5 operon also contains several accessory genes that are predicted to be involved in different stages of biosynthesis of the enzyme. In this work, deletions in the hydF, hydG, and hydH genes have been constructed. The hydF gene encodes a protein related to Ralstonia eutropha HoxO, which is known to interact with the small subunit of a [NiFe]-hydrogenase. HydG is predicted to be a fusion of the R. eutropha HoxQ and HoxR proteins, both of which have been implicated in the biosynthesis of an O2-tolerant hydrogenase, and HydH is a homologue of R. eutropha HoxV, which is a scaffold for [NiFe] cofactor assembly. It is shown here that HydG and HydH play essential roles in Hyd-5 biosynthesis. Hyd-5 can be isolated and characterized from a ΔhydF strain, indicating that HydF may not play the same vital role as the orthologous HoxO. This study, therefore, emphasises differences that can be observed when comparing the function of hydrogenase maturases in different biological systems