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NASA NTRS2007

Improving Robotic Operator Performance Using Augmented Reality

The Special Purpose Dexterous Manipulator (SPDM) is a two-armed robot that functions as an extension to the end effector of the Space Station Robotics Manipulator System (SSRMS), currently in use on the International Space Station (ISS). Crew training for the SPDM is accomplished using a robotic hardware simulator, which performs most of SPDM functions under normal static Earth gravitational forces. Both the simulator and SPDM are controlled from a standard robotic workstation using a laptop for the user interface and three monitors for camera views. Most operations anticipated for the SPDM involve the manipulation, insertion, and removal of any of several types of Orbital Replaceable Unit (ORU), modules which control various ISS functions. Alignment tolerances for insertion of the ORU into its receptacle are 0.25 inch and 0.5 degree from nominal values. The pre-insertion alignment task must be performed within these tolerances by using available video camera views of the intrinsic features of the ORU and receptacle, without special registration markings. Since optimum camera views may not be available, and dynamic orbital lighting conditions may limit periods of viewing, a successful ORU insertion operation may require an extended period of time. This study explored the feasibility of using augmented reality (AR) to assist SPDM operations. Geometric graphical symbols were overlaid on one of the workstation monitors to afford cues to assist the operator in attaining adequate pre-insertion ORU alignment. Twelve skilled subjects performed eight ORU insertion tasks using the simulator with and without the AR symbols in a repeated measures experimental design. Results indicated that using the AR symbols reduced pre-insertion alignment error for all subjects and reduced the time to complete pre-insertion alignment for most subjects.

Maida, James C.↗

DOE OSTI2023

Embedding ceramic components in metal structures with hybrid directed energy deposition

The combined benefit of both additive and subtractive manufacturing within the same gantry system enables hybrid directed energy deposition to create complex geometries with smooth surface finish and superior dimensional accuracy. Moreover, with layer-by-layer access to the structure during both the addition and subtraction of material, the insertion of components is now possible, assuming the components can survive the high temperatures associated with the subsequent metal deposition. Ceramic inserts are of interest for a variety of reasons including (1) to create complex interwoven ductile/brittle composites for ballistics or high-temperature applications or (2) to integrate high-temperature strain or temperature sensors protected within ceramic substrate subsumed into a larger metal structure. Here, in this work, stainless steel substrates were machined to create an internal cavity for the insertion of a ceramic component. During the investigation of several different over-the-ceramic deposition strategies, components were inserted, and different process sequences were allowed to continue to envelop the inserted ceramic with varying success. Unmelted powder was used to serve both as a thermal buffer and to provide a flush surface upon which the laser cladding could continue. Subsequent depositions were attempted with both dry and wet powder (addition of machining coolant to wet). The wet powder has previously been demonstrated to not significantly impact the mechanical properties of a final structure and provided a thermal barrier to protect the ceramic piece from the extreme temperatures of the final metal deposition. The wetting of the powder provided stability and minimized displacement caused by the powder flow from the laser cladding head. Finally, the use of an oblique angle for laser cladding allowed for the redirection of some fraction of the introduced thermal energy away from the ceramic component and, consequently, improved the survival of the ceramic inserts. With this combination of techniques, ceramic inserts survived full embedding within a 3D-printed stainless steel structure.

36 MATERIALS SCIENCE↗

DOE OSTI2024

Linear Free Energy Relationships Associated with Hydride Transfer From [(6,6'-R 2 -bpy)Re(CO) 3 H]: A Cautionary Tale in Identifying Hydrogen Bonding Effects in the Secondary Coordination Sphere

Here, six rhenium hydride complexes, [(6,6'-R 2 -bpy)Re(CO) 3 H] (bpy = 2,2'-bipyridine, R = OEt, OMe, NHMe, Me, F, Br), were synthesized. These complexes insert CO 2 to form rhenium formate complexes of the type [(6,6'-R 2 -bpy)Re(CO) 3 {OC(O)H}]. All the rhenium formate species were characterized using X-ray crystallography, which revealed that the bpy ligand is not coplanar with the metal coordination plane containing the two nitrogen donors of the bpy ligand but tilted. A solid-state structure of [(6,6'-Me 2 -bpy)Re(CO) 3 H] determined using MicroED also featured a tilted bpy ligand. The kinetics of CO 2 insertion into complexes of the type [(6,6'-R 2 -bpy)Re(CO) 3 H] were measured experimentally and the thermodynamic hydricities of [(6,6'-R 2 -bpy)Re(CO) 3 H] species were determined using theoretical calculations. A Brønsted plot constructed using the experimentally determined rate constants for CO 2 insertion and the calculated thermodynamic hydricities for [(6,6'-R 2 -bpy)Re(CO) 3 H] revealed a linear free energy relationship (LFER) between thermodynamic and kinetic hydricity. This LFER is different to the previously determined relationship for CO 2 insertion into complexes of the type [(4,4'-R 2 -bpy)Re(CO) 3 H]. At a given thermodynamic hydricity, CO 2 insertion is faster for complexes containing a 6,6'-substituted bpy ligand. This is likely in part due to the tilting observed for systems with 6,6'-substituted bpy ligands. Notably, the 6,6'-(NHMe) 2 -bpy ligand could in principle stabilize the transition state for CO 2 insertion via hydrogen bonding. This work shows that if only the rate of CO 2 insertion into [(6,6'-(NHMe) 2 -bpy)Re(CO) 3 H] is compared to [(4,4'-R 2 -bpy)Re(CO) 3 H] systems, the increase in rate could be easily attributed to hydrogen bonding, but in fact all 6,6'-substituted systems lead to faster than expected rates.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

Collar Properties and Measurement Time Confer Minimal Bias Overall on Annual Soil Respiration Estimates in a Global Database

Abstract Measuring the soil‐to‐atmosphere carbon dioxide (CO 2 ) flux (soil respiration, R S ) is important to understanding terrestrial carbon balance and to forecasting climate change. Such measurements are frequently made using measurement collars permanently inserted into the soil surface. However, differences in measurement duration and frequency, as well as collar properties, may lead to biases in the estimation of annual R S . Using a newly updated global R S database (SRDB‐V5), we investigated the annual R S bias associated with five methodological factors: collar height, collar coverage area, collar insertion depth, measurement duration, and measurement frequency. We found that annual R S was negatively correlated with collar insertion depth, consistent with the idea that collar insertion cuts roots and thus reduces R S . Annual R S was also negatively related with collar height and collar coverage area, perhaps because uniform head‐space mixing is difficult to achieve in larger volume chambers; however, these effects were quantitatively small (bias of ~2% to 10% of mean R S ). We found no correlation of measurement duration or measurement frequency with annual R S . These findings suggest that variation in R S methodology generally introduces minimal bias overall. Therefore, compilations of minimally adjusted annual R S measurements provide a reliable resource for synthesis studies, global annual R S modeling, and investigation of how soil carbon responds to climate change.

Jian, Jinshi↗

NASA NTRS1998

Evaluation of dual-tip micromanometers during 21-day implantation in goats

Investigative research efforts using a cardiovascular model required the determination of central circulatory haemodynamic and arterial system parameters for the evaluation of cardiovascular performance. These calculations required continuous beat-to-beat measurement of pressure within the four chambers of the heart and great vessels. Sensitivity and offset drift, longevity, and sources of error for eight 3F dual-tipped micromanometers were determined during 21 days of implantation in goats. Subjects were instrumented with pairs of chronically implanted fluid-filled access catheters in the left and right ventricles, through which dual-tipped (test) micromanometers were chronically inserted and single-tip (standard) micromanometers were acutely inserted. Acutely inserted sensors were calibrated daily and measured pressures were compared in vivo to the chronically inserted sensors. Comparison of the pre- and post-gain calibration of the chronically inserted sensors showed a mean sensitivity drift of 1.0 +/- 0.4% (99% confidence, n = 9 sensors) and mean offset drift of 5.0 +/- 1.5 mmHg (99% confidence, n = 9 sensors). Potential sources of error for these drifts were identified, and included measurement system inaccuracies, temperature drift, hydrostatic column gradients, and dynamic pressure changes. Based upon these findings, we determined that these micromanometers may be chronically inserted in high-pressure chambers for up to 17 days with an acceptable error, but should be limited to acute (hours) insertions in low-pressure applications.

Non-NASA Center↗

NASA NTRS2012

The DECLIC Research Facility - a Fertile Platform for NASA/CNES Scientific Collaboration

The DECLIC (Device for the Study of Critical Liquids and Crystalization) Facility was launched to the International Space Station (ISS) on Shuttle flight 17-A (August 2009) and has been in service for a little over three years. Activity from the three originally planned investigations, the HTI (High Temperature Insert) investigation, the ALI (Alice Like Insert) investigation and the DSI (Directional Solidication Insert) investigation has led to fruitful collaborations among a team of scientists, sponsored by NASA and CNES, to extend the utility of the inserts and the breadth of science beyond its initial scope. These follow-on investigations plan to use inserts that have been returned to earth for refurbishment, two of which (i.e., HTI-R and DSI-R) simply entail changing the test sample and the third (i.e., ALI-R) entails a slight hardware modication to allow for precise changes in sample volume. The first investigation, the Supercritical Water Mixture (SCWM) experiment, uses the refurbished HTI-R, which will accommodate a dilute aqueous mixture of Na2SO4 -0.5% w. This investigation will extend earlier observations of pure water at near-critical conditions. The second experiment uses a modified insert, the DSI-R, with a different concentration of succinonitrile-camphor than the original flight sample. This will allow, among other objectives, a detailed study of dendritic sidebranch formation in extended three-dimensional arrays, with the goal of elucidating whether noise amplication and/or a deterministic limit cycle is the main cause of sidebranch formation. The final experiment, the ALI-R, uses a sample cell with variable density to allow for additional observations of thermo-physical properties on SF6 at near critical conditions. The presentation will provide a discussion of the DECLIC facility's hardware, its modied inserts, and an overview of the extended science that will be achieved through these collaborative activities.

Hicks, Michael C.↗

DOE OSTI2021

Configurable vehicle seat and method therefor

A conformal seat insert for an adjustable vehicle seat, the conformal seat insert comprising an insert frame, the insert frame having one or more insert reference features for locating the insert frame on a seat frame of the vehicle seat in a predetermined position relative to the seat frame, and a contoured support surface formed in the insert frame, the contoured support surface having a crew reference point corresponding to an acromial point of a shoulder of a seat occupant of the adjustable vehicle seat, the crew reference point being a datum from which an eye position of the seat occupant is established relative to instrumentation of an aerospace vehicle in which the adjustable vehicle seat is located.

McCarley, Michael C.↗

DOE OSTI2024

Reactivity of [Tism Pr i Benz ]MgH and [Tism Pr i Benz ]MgMe towards Carbonyl Compounds: Access to Terminal Alkoxide and Enolate Complexes

The hydride and methyl compounds [Tism Pr i Benz ]MgH and [Tism Pr i Benz ]MgMe undergo insertion of the carbonyl moieties of non-enolizable aldehydes and ketones such as PhCHO and Ph 2 CO into the Mg–H and Mg–Me bonds to form alkoxide compounds, namely [Tism Pr i Benz ]MgOCH 2 Ph, [Tism Pr i Benz ]MgOCHPh 2 , [Tism Pr i Benz ]MgMOCH(Me)Ph and [Tism Pr i Benz ]MgOCMePh 2 . In contrast to the insertion of the carbonyl moiety, the reactions of the enolizable ketones Me 2 CO and PhC(O)Me with [Tism Pr i Benz ]MgMe afford the enolate complexes, [Tism Pr i Benz ]MgOC(Me)=CH 2 and [Tism Pr i Benz ]MgOC(Ph)=CH 2 . The formation of [Tism Pr i Benz ]MgOC(Me)=CH 2 is of note because methyl Grignard reagents preferentially react with acetone to form t-butoxide derivatives. The hydride compound, [Tism Pr i Benz ]MgH, also reacts with acetone to yield the enolate compound, [Tism Pr i Benz ]MgOC(Me)=CH 2 , but while the overall transformation is similar to that of the methyl derivative, [Tism Pr i Benz ]MgMe, the enolate compound is not the initially formed product. Specifically, acetone undergoes preferential insertion into the Mg–H bond to generate the corresponding alkoxide, [Tism Pr i Benz ]MgOPr i , which subsequently converts to the respective enolate in the presence of excess acetone. Furthermore, the relative ability of the hydride and methyl compounds to undergo insertion of carbonyl compounds into the Mg–H and Mg–Me bonds has been addressed computationally, which indicates that the barrier for insertion of the carbonyl group into the Mg–H bond is lower than that for insertion into the Mg–Me bond. The molecular structures of [Tism Pr i Benz ]MgOCH 2 Ph, [Tism Pr i Benz ]MgOCHPh 2 , [Tism Pr i Benz ]MgOCMePh 2 , [Tism Pr i Benz ]MgOC(Me)=CH 2 and [Tism Pr i Benz ]MgOC(Ph)=CH 2 have been determined by X-ray diffraction.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS2005

Experimental Investigation of Oscillatory Flow Pressure and Pressure Drop Through Complex Geometries

A series of experiments have been performed to investigate the oscillatory flow pressure and pressure drop through complex geometries. These experiments were conducted at the CSU-SLRE facility which is a horizontally opposed, two-piston, single-acting engine with a split crankshaft driving mechanism. Flow through a rectangular duct, with no insert (obstruction), was studied first. Then four different inserts were examined: Abrupt, Manifold, Diverging Short and Diverging Long. The inserts were mounted in the center of the rectangular duct to represent different type of geometries that could be encountered in Stirling machines. The pressure and pressure drop of the oscillating flow was studied for: 1) different inserts, 2) different phase angle between the two pistons of the engine (zero, 90 lead, 180, and 90 lag), and 3) for different piston frequencies (5, 10, 15, and 20 Hz). It was found that the pressure drop of the oscillatory flow increases with increasing Reynolds number. The pressure drop was shown to be mainly due to the gas inertia for the case of oscillatory flow through a rectangular duct with no insert. On the other hand, for the cases with different inserts into the rectangular duct, the pressure drop has three sources: inertia, friction, and local losses. The friction pressure drop is only a small fraction of the total pressure drop. It was also shown that the dimensionless pressure drop decreases with increasing kinetic Reynolds number.

Ibrahim, Mounir B.↗

NASA NTRS2006

Repairing Fractured Bones by Use of Bioabsorbable Composites

A proposed method of surgical repair of fractured bones would incorporate recent and future advances in the art of composite materials. The composite materials used in this method would be biocompatible and at least partly bioabsorbable: that is, during the healing process following surgery, they would be wholly or at least partly absorbed into the bones and other tissues in which they were implanted. Relative to the traditional method, the proposed method would involve less surgery, pose less of a risk of infection, provide for better transfer of loads across fracture sites, and thereby promote better healing while reducing the need for immobilization by casts and other external devices. One requirement that both the traditional and proposed methods must satisfy is to fix the multiple segments of a broken bone in the correct relative positions. Mechanical fixing techniques used in the traditional method include the use of plates spanning the fracture site and secured to the bone by screws, serving of wire along the bone across the fracture site, insertion of metallic intramedullary rods through the hollow portion of the fractured bone, and/or inserting transverse rods through the bone, muscle, and skin to stabilize the fractured members. After the bone heals, a second surgical operation is needed to remove the mechanical fixture(s). In the proposed method, there would be no need for a second surgical operation. The proposed method is based partly on the observation that in the fabrication of a structural member, it is generally more efficient and reliable to use multiple small fasteners to transfer load across a joint than to use a single or smaller number of larger fasteners, provided that the stress fields of neighboring small fasteners do not overlap or interact. Also, multiple smaller fasteners are more reliable than are larger and fewer fasteners. However, there is a trade-off between structural efficiency and the cost of insertion time and materials. The proposed method is further based partly on the conjecture that through-the-thickness reinforcements could be excellent for fixing bone segments for surgical repair. The through-the-thickness reinforcements would superficially resemble nails in both form and function. Denoted small-diameter rods (SDRs) to distinguish them from other narrow rods, these reinforcements would be shot or otherwise inserted through adjacent segments of fractured bone to fix them in their correct relative positions (see figure). Shot insertion would be effected by use an applicator that would amount to a miniaturized and highly refined version of the pneumatic guns often used in carpentry to drive nails and brads. The applicator, envisioned to be about the size of a ball-point-pen, would be driven by pressurized carbon dioxide. To further promote stabilization of the segments, layers of bone glue could be applied to the fracture surfaces prior to insertion of the SDRs. The bone glue could be therapeutically loaded with chemicals to promote growth of bone and fight infection

Farley, Gary L.↗

NASA NTRSDate not supplied

Uranus Flagship-class Orbiter and Probe Using Aerocapture

Exploration of the Ice Giants, especially Uranus, via orbiter and atmospheric probes, is required to answer pressing science questions that have been raised in the latest National Academies of Sciences Planetary Decadal Survey. Since the Ice Giants are the farthest planets from Earth, traditional fully-propulsive orbit insertion missions have transit times to the planetary bodies bordering 13-15 years and require a large amount of propellant (wet mass percentages of around 60-70%) for the orbit insertion maneuver, leaving less mass for the scientific payload and a planetary probe. Aerocapture uses aerodynamic forces generated by flight within a planetary atmosphere to decelerate and achieve orbit insertion. Aerocapture has been considered for several past missions but it has not been demonstrated. However, recent developments in thermal protection systems (TPS), guidance and control (G&C), and interplanetary navigation capabilities show the potential for using rigid, heritage entry vehicle configurations already flown at other planetary bodies for Ice Giants aerocapture. Aerocapture can robustly deliver spacecraft to Ice Giant orbits, while substantially increasing on-orbit payload mass (more than 40%) that can be used for a robust atmospheric entry probe. Additionally, the aerocapture maneuver would reduce the interplanetary transit time by 2-5 years (15-30%) relative to fully-propulsive orbit insertion. Recent work has shown that a flagship-class mission can be conducted in a shorter time than fully-propulsive missions if using aerocapture. This paper will consider the merits of including aerocapture as the orbit-insertion mechanism for a Uranus mission. Specifically, the implications of aerocapture orbit insertion for in-situ atmospheric probes will be discussed. The Uranus Orbiter and Probe concept mission study [3] is considered as the potential payload. Results from a recent NASA Space Technology Mission Directorate (STMD)-funded activity that is designing an aerocapture mission for a Uranus orbiter will be presented.

Soumyo Dutta↗

NASA NTRSDate not supplied

Uranus Flagship-class Orbiter and Probe Using Aerocapture

Soumyo Dutta↗

NASA NTRS2024

Aerocapture: An Enabling Technology for Flagship-Class Uranus Orbiter and Probe Mission

Introduction: The current decadal survey published by the National Academies of Sciences has informed National Aeronautics and Space Administration (NASA) to prioritize the study of the Ice Giants, especially Uranus. To gather the required data that addresses the science questions raised in this survey, a mission to Uranus with an orbiter and atmospheric probe must be designed. The Uranus Orbiter and Probe (UOP) study, which the survey identified as the flagship mission of this decade, proposes a 2031 launch to take advantage of a Jupiter fly-by and utilizes a fully propulsive orbit insertion design with an Earth-to-Uranus transit times ranging from 13 to 15 years. This fully propulsive orbit insertion at Uranus will be very fuel expensive (wet mass percentages of around 60-70\%) thereby leaving less mass for the scientific payload and additional planetary probes. In addition, scientists are more interested in visiting Uranus before 2049, when the Spring Equinox will occur, as it allows studying Uranus seasons not seen during Voyager 2's flyby in 1987. A NASA Flagship-Class mission would require at least 10 years of lead time prior to launch thereby making the 2031 launch to take advantage of the Jupiter fly-by extremely challenging. The consequence of missing the Jupiter fly-by and launching in the late 2030s is the challenge of a fully-propulsive mission like UOP to have a feasible alternative interplanetary trajectory that reaches Uranus before 2050. As an alternative, to address the shortcomings of the fully propulsive mission, one can design a mission to Uranus using aerocapture. What is aerocapture: Aerocapture is an atmospheric maneuver that uses aerodynamic forces generated by flight within a planetary atmosphere to decelerate and achieve orbit insertion. Using aerocapture allows one to provide the change in velocity (Delta V) needed to slow down from the approach hyperbolic trajectory to achieve the desired captured orbit around the target planet using aerodynamic forces of the vehicle (lift and drag). Using aerodynamic forces instead of fully-propulsive maneuvers results in significant savings in the fuel. Furthermore, aerocapture can also allow one to consider interplanetary trajectories with higher approach hyperbolic velocities, thus reducing the mission transit times. Aerocapture as an enabling technology: To use aerocapture as an enabling technology for the Uranus exploration mission, one would require an integrated system-level design, including a Thermal Protection System (TPS), hardware needed for aerodynamic modulation, and autonomous Guidance, Navigation, and Control (GNC) systems. Aerocapture has yet to be demonstrated, despite considering it for several past missions. Recent advancements in TPS and GNC capabilities show the potential for using rigid, heritage entry vehicle configurations already flown at other planetary bodies for Uranus aerocapture. Aerocapture can be a robust technology that can deliver spacecraft to Uranus science orbits while substantially increasing on-orbit payload mass (more than 40\%) that can enable robust atmospheric entry probes. Additionally, the aerocapture maneuver would reduce the interplanetary transit time by 2–5 years (15-30\%) relative to fully-propulsive orbit insertion. Recent work has shown that one can conduct a flagship-class mission in a shorter period than fully-propulsive missions if using aerocapture. What does aerocapture bring in for a Uranus Mission: Using aerocapture for a Uranus orbiter and atmospheric probe mission can provide one with considerable propellant savings. Spacecraft in interplanetary trajectories to Uranus typically need an Delta V in orders of kilometers per second to insert into science orbit. One would require thousands of kilograms of fuel to achieve such a Delta V using a traditional fully propulsive maneuver, leaving less mass for payload during the mission launch. Aerocapture can reduce the propulsion needs by dissipating energy in the sizable atmosphere of Uranus without a significant mass increase due to the need for an aeroshell. One can use the mass savings achieved using the aerocapture to reduce the launch vehicle requirements. In addition, one can also have additional science instruments on the orbiter or create a robust instrumentation suite on an atmospheric probe that can significantly increase the science outcome of the Uranus exploration mission. Furthermore, since aerocapture performance is relatively insensitive to increases in hyperbolic excess velocity, one can design the interplanetary trajectory to arrive at Uranus faster, reducing the interplanetary transit time and operations cost. All these savings, achieved using aerocapture, could help fit a larger class mission, such as the Uranus mission within a smaller capital, e.g., a Flagship-class orbiter mission in a New Frontiers class capital. Summary: This talk will provide an overview of how aerocapture can enable the Uranus exploration mission. Specifically, this talk will discuss the latest advancements made in the Uranus aerocapture study, such as investigating interplanetary trajectories with higher hyperbolic approach velocities and their implications on the aero heating and the TPS design, incorporating FNPAG (an advanced numerical-predictor guidance) and comparison of multiple navigation approaches. In addition, this talk will focus on mechanical design that can house more than one atmospheric probe and the six degrees of freedom simulation of aerocapture at Uranus. Findings from a recent NASA Space Technology Mission Directorate (STMD)-funded activity studying the aerocapture as an enabling technology for a Uranus orbiter will be presented. Using the science payload recommended by the Decadal Survey for Uranus exploration, this work shows many improvements over the baseline fully-propulsive mission. These improvements include a shorter cruise phase, flexibility in launch opportunities late into the 2030s while reaching Uranus before the 2050 equinox for the desired science opportunities, and lower propellant mass needs. This talk will highlight how aerocapture can be utilized for Uranus science orbit insertion using a lower-risk, heritage entry vehicle configuration used extensively as a Mars entry, descent, and landing vehicle. Furthermore, this talk will explore how the demonstration of aerocapture at Earth can benefit the aerocapture-enabled Uranus mission.

Aerocapture↗

NASA NTRS1993

Phenotypic characterization of xpr, a global regulator of extracellular virulence factors in Staphylococcus aureus

We recently described a Tn551 insertion in the chromosome of Staphylococcus aureus S6C that resulted in drastically reduced expression of extracellular lipase (M. S. Smeltzer, S. R. Gill, and J. J. Iandolo, J. Bacteriol. 174:4000-4006, 1992). The insertion was localized to a chromosomal site (designated omega 1058) distinct from the lipase structural gene (geh) and the accessory gene regulator (agr), both of which were structurally intact in the lipase-negative (Lip-) mutants. In this report, we describe a phenotypic comparison between strains S6C, a hyperproducer of enterotoxin B; KSI9051, a derivative of S6C carrying the Tn551 insertion at omega 1058; ISP546, an 8325-4 strain that carries a Tn551 insertion in the agr locus; and ISP479C, the parent strain of ISP546 cured of the Tn551 delivery plasmid pI258repA36. Compared with their respective parent strains, ISP546 and KSI9051 produced greatly reduced amounts of lipase, alpha-toxin, delta-toxin, protease, and nuclease. KSI9051 also produced reduced amounts of staphylococcal enterotoxin B. Coagulase production was increased in ISP546 but not in KSI9051. Using a mouse model, we also demonstrated that ISP546 and KSI9051 were far less virulent than ISP479C and S6C. We have designated the genetic element defined by the Tn551 insertion at omega 1058 xpr to denote its role as a regulator of extracellular protein synthesis. We conclude that xpr and agr are similar and possibly interactive regulatory genes that play an important role in pathogenesis of staphylococcal disease.

NASA Discipline Cell Biology↗

DOE OSTI2023

A cellular selection identifies elongated flavodoxins that support electron transfer to sulfite reductase

Flavodoxins (Flds) mediate the flux of electrons between oxidoreductases in diverse metabolic pathways. To investigate whether Flds can support electron transfer to a sulfite reductase (SIR) that evolved to couple with a ferredoxin, we evaluated the ability of Flds to transfer electrons from a ferredoxin-NADP reductase (FNR) to a ferredoxin-dependent SIR using growth complementation of an Escherichia coli strain with a sulfur metabolism defect. We show that Flds from cyanobacteria complement this growth defect when coexpressed with an FNR and an SIR that evolved to couple with a plant ferredoxin. When we evaluated the effect of peptide insertion on Fld-mediated electron transfer, we observed a sensitivity to insertions within regions predicted to be proximal to the cofactor and partner binding sites, while a high insertion tolerance was detected within loops distal from the cofactor and within regions of helices and sheets that are proximal to those loops. Bioinformatic analysis showed that natural Fld sequence variability predicts a large fraction of the motifs that tolerate insertion of the octapeptide SGRPGSLS. In conclusion, these results represent the first evidence that Flds can support electron transfer to assimilatory SIRs, and they suggest that the pattern of insertion tolerance is influenced by interactions with oxidoreductase partners.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Transposon sequencing analysis of Bradyrhizobium diazoefficiens 110 spc4

Bradyrhizobium diazoefficiens USDA110 is one of the most effective nitrogen-fixing symbionts of soybeans. Here we carried out a large-scale transposon insertion sequencing (Tn-seq) analysis of strain Bd110spc4, which is derived from USDA110, with the goal of increasing available resources for identifying genes crucial for the survival of this plant symbiont under diverse conditions. We prepared two transposon (Tn) insertion libraries of Bd110spc4 with 155,042 unique Tn insertions when the libraries were combined, which is an average of one insertion every 58.7 bp of the reference USDA110 genome. Application of bioinformatic filtering steps to remove genes too small to be expected to have Tn insertions, resulted in a list of genes that were classified as putatively essential. Comparison of this gene set with genes putatively essential for the growth of the closely related alpha-proteobacterium, Rhodopseudomonas palustris, revealed a small set of five genes that may be collectively essential for closely related members of the family Bradyrhizobiaceae. This group includes bacteria with diverse lifestyles ranging from plant symbionts to animal-associated species to free-living species.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Engineered mRNA–ribosome fusions for facile biosynthesis of selenoproteins

Ribosomes are often used in synthetic biology as a tool to produce desired proteins with enhanced properties or entirely new functions. However, repurposing ribosomes for producing designer proteins is challenging due to the limited number of engineering solutions available to alter the natural activity of these enzymes. In this study, we advance ribosome engineering by describing a novel strategy based on functional fusions of ribosomal RNA (rRNA) with messenger RNA (mRNA). Specifically, we create an mRNA–ribosome fusion called RiboU, where the 16S rRNA is covalently attached to selenocysteine insertion sequence (SECIS), a regulatory RNA element found in mRNAs encoding selenoproteins. When SECIS sequences are present in natural mRNAs, they instruct ribosomes to decode UGA codons as selenocysteine (Sec, U) codons instead of interpreting them as stop codons. This enables ribosomes to insert Sec into the growing polypeptide chain at the appropriate site. Our work demonstrates that the SECIS sequence maintains its functionality even when inserted into the ribosome structure. As a result, the engineered ribosomes RiboU interpret UAG codons as Sec codons, allowing easy and site-specific insertion of Sec in a protein of interest with no further modification to the natural machinery of protein synthesis. To validate this approach, we use RiboU ribosomes to produce three functional target selenoproteins in Escherichia coli by site-specifically inserting Sec into the proteins’ active sites. Overall, our work demonstrates the feasibility of creating functional mRNA–rRNA fusions as a strategy for ribosome engineering, providing a novel tool for producing Sec-containing proteins in live bacterial cells.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Structures of vertebrate R2 retrotransposon complexes during target-primed reverse transcription and after second-strand nicking

R2 retrotransposons are site-specific eukaryotic non–long terminal repeat retrotransposons that copy and paste into gene loci encoding ribosomal RNAs. Recently, we demonstrated that avian A-clade R2 proteins achieve efficient and precise insertion of transgenes into their native safe-harbor loci in human cells. The features of A-clade R2 proteins that support gene insertion are not well characterized. Here, we report high-resolution cryo–electron microscopy structures of two vertebrate A-clade R2 proteins at the initiation of target-primed reverse transcription and after cDNA synthesis and second-strand nicking. Using biochemical and cellular assays, we illuminate the basis for high selectivity of template use and unique roles for each of the three zinc-finger domains in nucleic acid recognition. Reverse transcriptase active site architecture is reinforced by an unanticipated insertion motif specific to vertebrate A-clade R2 proteins. Our work provides the first insights into A-clade R2 protein structure during gene insertion and may enable future improvement and adaptation of R2-based systems for precise transgene insertion.

Science & Technology - Other Topics↗