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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Advancing Protein Display on Bacterial Spores through an Extensive Survey of Coat Components

The profound stability of bacterial spores makes them a promising platform for biotechnological applications like biocatalysis, bioremediation, drug delivery, etc. However, though the Bacillus subtilis spore is composed of >40 types of proteins, only ∼12 have been explored as fusion carriers for protein display. Here, we assessed the suitability of 33 spore proteins (SPs) as enzyme display carriers by direct allele tagging at native genomic loci. Of the 33 SPs investigated, 26 formed functional fusions with β-glucuronidase (GUS)─a ∼272 kDa homotetramer. This almost triples the number of SPs assessed for enzyme display and doubles the number of functional fusions documented in the literature. We quantitatively assessed 1) SP promoter activation dynamics, 2) GUS activity on spores, 3) surface availability, and 4) protection from thermal and proteolytic degradation. Multicopy expression and pairwise coexpression of the most promising SP-GUS fusions highlighted the complexity of spore structure/assembly and the difficulty in predicting compatibility between different SP fusions. We also assessed the suitability of engineered spores to degrade PET (polyethylene terephthalate) films and found that surface-exposed SPs were most effective. Beyond the broad survey, a key outcome of our work was the identification of SscA (small spore coat assembly protein A) as an effective spore display carrier. SscA supported enzyme activity at least 4-fold higher than any other SP, including the well-established anchor, CotY. We attribute this to its promoter, which demonstrated early and sustained activation relative to other SPs and its small size (∼3 kDa), which likely minimally interferes with enzyme folding, oligomerization, and activity. Labeling and genetic studies, its hydrophobic nature, and low surface availability suggest that SscA assembles within the inner spore coat, which makes it stabilizing and suitable for many biocatalytic applications. Overall, this work serves as a knowledge base to advance the biotechnological utility of B. subtilis spores.

Bacillus subtilis↗
NASA NTRS1973

Effect of simulated lunar impact on the survival of bacterial spores.

In order to test the effect of impact on organisms, the survival of bacterial spores after being propelled at high velocity in Pyrex and plastic beads into crushed basalt was measured. The beads were fired into sterilized canisters by both a conventional powder and a light gas gun. Results indicate that at the minimum (2.4 km/sec) lunar capture velocity, the number of colony forming units (CFUs) decreased by five orders of magnitude, and at 5.5 km/sec, statistically a more probable capture velocity, no CFUs were found. The decrease in CFUs observed with increasing velocity indicates that the spores were most probably killed by the impact.

Whitfield, O.↗
NASA NTRS1978

Thermal resistance of naturally occurring airborne bacterial spores

Simulation of a heat process used in the terminal dry-heat decontamination of the Viking spacecraft is reported. Naturally occurring airborne bacterial spores were collected on Teflon ribbons in selected spacecraft assembly areas and subsequently subjected to dry heat. Thermal inactivation experiments were conducted at 105, 111.7, 120, 125, 130, and 135 C with a moisture level of 1.2 mg of water per liter. Heat survivors were recovered at temperatures of 135 C when a 30-h heating cycle was employed. Survivors were recovered from all cycles studied and randomly selected for identification. The naturally occurring spore population was reduced an average of 2.2 to 4.4 log cycles from 105 to 135 C. Heating cycles of 5 and 15 h at temperature were compared with the standard 30-h cycle at 111.7, 120, and 125 C. No significant differences in inactivation (alpha = 0.05) were observed between 111.7 and 120 C. The 30-h cycle differs from the 5- and 15-h cycles at 125 C. Thus, the heating cycle can be reduced if a small fraction (about 0.001 to 0.0001) of very resistant spores can be tolerated.

Puleo, J. R.↗
NASA NTRS1973

An observation about the relative hardiness of bacterial spores and planetary quarantine

Planetary quarantine objectives are shown to be critically dependent on the deviation in the actual decimal-reduction-time or D values (i.e., the time necessary to reduce the population to one-tenth of its original value) of organisms on spacecraft from the values chosen for spacecraft sterilization that have been selected conservatively relative to defined experimental procedures and bacterial spore stocks. New data indicate that these D values are not conservative when compared with those of naturally occurring organisms. The possible implications of these new data for planetary quarantine are analyzed.

Trauth, C. A., Jr.↗
NASA NTRS1973

Release of Bacterial Spores from the Inner Walls of a Stainless Steel Cup Subjected to Thermal Stresses and Mechanical Shock

The release and fallout of particulates from surfaces afforded thermal or impact stress is of concern for control of contamination of Mars from planetary landing vehicles. A metal vessel contaminated by aerosols of spores was used as a model system and the fallout of spores as affected by various mechanisms was examined. Thermal stresses simulating those expected on the Mars lander dislodged approximately .01% of the aerosol deposited surface burden as did a landing shock of 8 to 10G deceleration. Spores imprinted by finger or swab contact yielded similar results. In all cases where repeated cycling of temperature, motion, or shock were employed the majority of fallout occurred in the first cycle. Particles released from the surface were predominantly in the size range 1 to 5 microns.

Wolochow, H.↗
NASA NTRS1992

Long-term exposure of bacterial spores to space

With the NASA mission of the Long Duration Exposure Facility (LDEF), the authors have obtained the opportunity to expose Bacillus subtilis spores for nearly six years to the space environment and to analyze their responses after retrieval. The experiment was mounted onto a side tray of LDEF facing space. Data shows that the chances of microorganisms surviving in free space will be greatly increased by adequate shielding against solar ultraviolet light.

Horneck, G.↗
NASA NTRS1972

Services provided in support of the planetary quarantine requirements of the National Aeronautics and Space Administration

Heat studies with the highly resistant bacterial spore isolated from Cape Kennedy soil were continued, and the D130C was determined. The interior surfaces of the command module of the Apollo 17 spacecraft were studied for microbial contamination during assembly and testing. The thermal resistance of naturally occurring airborne bacterial spores was determined, using the heating times of 2, 4, 6, and 8 hr. at 125 C. The evaluation of a terminal sterilization process for unmanned lander spacecraft is also continuing.

Favero, M. S.↗
NASA NTRS1984

Chemical and biological evolution in space

The formation of organic molecules in interstellar dust grains and the survival of bacterial spores in a space environment are examined, summarizing the results of experimental investigations and theoretical calculations. The interstellar medium is characterized; the production of organic yellow-stuff residues on the mantles of dust grains in molecular clouds by UV radiation is explained; an IR absorption spectrum toward a Galactic-center source is shown to confirm the presence of such residues; and the mass of such abiotically produced complex organic molecules in the Galaxy is estimated as at least 10 to the 7th solar mass, or 0.1 percent of the mass in the Galaxy. This finding is considered significant for the prebiotic chemistry of the earth, since comets containing large amounts of interstellar dust may have impacted the earth many times during comet showers in the early solar system. UV-irradiation experiments on bacterial spores show that initial exposures to solar UV, not interstellar survival, is the main factor rendering theries of panspermia questionable.

Greenberg, J. M.↗
DOE OSTI2025

Degradable Biocomposite Thermoplastic Polyurethanes

In this project, the team developed tough and degradable biocomposite thermoplastic polyurethanes (TPUs) by incorporating bacterial spores into TPUs as a biofunctional living filler. The team screened various bacteria and selected the Bacillus subtilis ATCC 6633 strain as the final candidate, primarily due to its genomic availability, sporulation ability and TPU assimilation activity. The heat-shock tolerance of ATCC 6633 spores was further improved through evolutionary engineering via Adaptive Laboratory Evolution (ALE), demonstrating a 17.7-fold enhanced germination efficiency post heat-shock treatment compared to the wild-type strain (WT). The team fabricated biocomposite TPUs by incorporating lyophilized powder of heat-shock tolerized (HST) spores during the hot melt extrusion (HME) of TPU at 135 °C. The baseline TPU used in this project is a commercially available soft-grade TPU (BCF45) manufactured by BASF. Colony forming unit (CFU) assays quantified that WT and HST spores in the TPU matrix retained approximately 20% and 100% survivability, respectively, after HME. Tensile testing demonstrated that the spores behaved as a polymer-reinforcing filler, positively affecting the overall tensile properties of the biocomposite TPU. For example, biocomposite TPU with WT and HST spores (BC TPU WT and BC TPU HST , respectively) exhibited up to 25% and 37% improved toughness, respectively, compared to TPU without spores. BC TPU HST showed remarkably improved disintegration in autoclaved compost (92% mass loss in 5 months), which simulated a microbially poor environment for TPU degradation. When compared to TPU without spores (44% mass loss in 5 months) the acceleration of degradation is marked. Respirometry confirmed that 72% of BC TPU HST was biomineralized into CO2 within 6 months, indicating that spores in the biocomposite TPU were germinated by utilizing nutrients in the autoclaved compost, facilitating TPU degradation at the end of the material's life. The team demonstrated the scale-up of biocomposite TPU fabrication using continuous extrusion and injection molding techniques. Processing conditions optimized in a lab-scale microcompounder were successfully transferred to a continuous extruder with a 30-fold increased throughput. Biocomposite TPUs prepared using these industry-relevant processes showed comparable toughness improvements to samples prepared in the lab-scale extruder. Excitingly, following compounding in the pilot-extruder the composite material could be injection molded, while retaining high spore viability and similar toughness improvements. The team also found that spores in biocomposite TPU served as antioxidants, preventing toughness decay during the recycled extrusion of BC TPU HST . Long-term storage tests over one year showed that the addition of spores had no negative effect on the longevity of the TPU. Furthermore, the team demonstrated the fabrication of spore-bearing biocomposite polymers with other polyesters such as PBAT, PLA, and PCL. We obtained promising preliminary data that showed overall toughness improvements for all polymers with spore addition. Finally, life cycle assessment (LCA) and techno-economic analysis (TEA) were carried out, which indicated minimal additional cost of fabrication. Overall, a tough and degradable biocomposite thermoplastic was successfully developed through this project, with all tasks completed successfully, achieving >100% of the objectives.

36 MATERIALS SCIENCE↗
NASA NTRS1973

Lyophilized spore dispenser

A lyophilized spore dispenser is provided which produces a finely divided, monoparticulate cloud of bacterial spores. The spores are contained within a tightly sealed chamber, and a turbulator orifice connected to an air supply source provides a jet of air which stirs up the spores and causes the spores to be suspended in eddy currents within the chamber. This air jet also produces a positive pressure within the chamber which forces the spores out of an injection orifice.

Jessup, A. D.↗
NASA NTRS1971

Planetary quarantine program

A quantitative means was developed to investigate the sensitivity of current spacecraft sterilization plans to variations in D-values. A quantitative expression was derived to represent the distribution of D-values among a population of naturally occurring organisms. An investigation was made of (1) the inactivation of both Bacillus subtilis var. niger spores and Cape Kennedy soil spores by gamma-radiation at room temperature in a nitrogen environment, and (2) the thermoradiation resistance of Cape Kennedy soil spores at elevated temperatures below 125 C. The relation between standard survival experiments with bacterial spores in soils and results obtained on spacecraft surfaces is discussed. Sporocidal properties of aqueous formaldehyde can be increased by elevating the temperature.

Source record↗
NASA NTRS1973

Ecology and thermal inactivation of microbes in and on interplanetary space vehicle components

An experimental sterilization facility was developed to simulate conditions that will be encountered during terminal sterilization of space vehicles. The system consists of a temperature controlled oven with a nitrogen gas stream containing a known concentration of water. Moisture analyzers are utilized to monitor the gas flowing over spore samples contained in the oven. In its original configuration, no provision was made for the control of water vapor during the sterilization cycle. Because moisture profoundly influences the thermal inactivation of bacterial spores, an upper limit for the moisture content in the gas used to sterilize the space vehicle was established (25% RH at 0 C STP). Accordingly, a controller was developed and installed to provide these conditions in the experimental sterilization facility.

Wimsatt, J. C.↗
DOE OSTI2025

Editorial: Structure and mechanism of microbial membrane active transporters

Membrane active transporters play essential roles in microbial physiology. They couple energy transduction to conformational changes that drive translocation of nutrients, substrates and ions, as well as molecular communication. The structure and function of microbial membrane active transporters are highly diverse. Typical examples include the primary active transporters in the ATP-binding cassette (ABC) superfamily (Thomas and Tampé, 2020; Davidson et al., 2008; Locher et al., 2002), the secondary active transporters in the Major Facilitator Superfamily (MFS) (Drew et al., 2021; Kaback and Guan, 2019), and the ligand-gated porins in the TonB-dependent transporter (TBDT) family (Klebba et al., 2021). As structural, proteogenomic, and computational methods advance, active transporters are increasingly recognized as dynamic molecular machines whose mechanisms can now be visualized and modeled with remarkable precision, building on decades of biochemical and biophysical discovery that established the foundations of this field. The transporter studies recruited in this Research Topic provide us with new insights into the field including structure-function of sugar transporters in yeast, structural prediction and classification of ABC complexes in Bacillus subtilis, Type VI Secretion System (T6SS) in Bacteroides fragilis, amino acids uptake in Escherichia coli and bacterial spore germination.

mechanism↗