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At least 361 records · Page 20

Culture-Independent Fungal Profiling for the International Space Station using Nanopore Sequencing: Method Development

Microbial monitoring of the International Space Station (ISS) environment is a crew health requirement that encompasses both bacterial and fungal identification. To achieve this currently, culture-based methods are used for sample collection, and these samples must be returned to the laboratory for analysis. The use of culture and the need for sample return to Earth results in a bias toward culturable organisms and causes a significant delay between sample collection and delivery of final data (weeks to months), respectively. Recently, advancements in molecular technology have aided a broad range of applications, including medical, industrial, and basic sciences. Additionally, increases in portability and ease-of-use of molecular platforms have provided point-of-use capabilities demonstrated by the miniPCR thermal cycler (miniPCR bio) and the MinION sequencer (Oxford Nanopore Technologies). Together, these devices have been applied to, and validated for, the identification of bacteria onboard the ISS. Building on this work, we have developed a spaceflight-compatible fungal workflow. Molecular-based fungal analysis is complicated by low biomass, difficult-to-lyse spores, debate regarding the region for taxonomic assignment, and the lack of bioinformatic pipelines and reference databases. To overcome these difficulties, primers yielding an ~ 2 Kb amplicon were validated against a wide range of ISS fungal isolates. The current spaceflight library preparation was substantially optimized, a bioinformatic pipeline was created, and refinements to the UNITE database were implemented. To compare this optimized method to the current culture-based standard, 30 sample sets (60 total swabs, two swabs held in tandem) were evaluated. Parallel fungal profiles were obtained between the two methods, with the culture-independent method revealing increased diversity. The addition of this method to the already established bacterial process fulfills the crew health identification requirement. Moreover, the implementation of this method onboard ISS will enhance our understanding of its unique fungal microbiome.

Hang N. Nguyen↗

Slippery Lubricant-Infused Silica Nanoparticulate Film Processing for Anti-Biofouling Applications

Microbial biofilm build-up in water distribution systems can pose a risk to human health and pipe material integrity. The impact is more devastating in space stations and to astronauts due to the isolation from necessary replacement parts and medical resources. As a result, there is a need for coatings to be implemented onto the inner region of the pipe to minimize the adherence and growth of biofilms. Lubricant-infused surfaces has been one such interesting material for anti-biofouling applications in which their slippery property promotes repellence to many liquids and thus prevents bacterial adherence. Textured and porous films are suitable substrate candidates to infuse and contain the lubricant. However, there is little investigation in utilizing a nanoporous thin film as the substrate material for lubricant infusion. A nanoporous film has high porosity within the structure which can promote greater lubricant infusion and retention. The implementation as a thin film structure aids to reduce material consumption and cost. In our study, we utilized a well-studied nanoporous thin film fabricated via layer-by-layer assembly of polycations and colloid silica and calcination for greater stability. The film was further functionalized to promote fluorinated groups upon the surface and pores of the film, which then could interact with a fluorinated lubricant. The pristine nanoporous film was characterized to determine its morphology, thickness, wettability, and porosity. The lubricant-infused film was then tested for its lubricant layer stability upon various washing conditions and its performance against bacterial biofilm adherence as a result of its slippery and thus anti-biofouling property.

biofilm↗

Biofilm Study Under Simulated Microgravity

The goal of this study was to understand biofilm formation under microgravity (µg), in support of biofilm mitigation efforts in exploration water recovery systems. The technical approach was to conduct a mass transfer and bacterial culture study under both simulated µg and ambient gravity. The aim was to correlate nutrient consumption to gene expression to better understand biofilm formation. A representative species of bacteria that is commonly cultured from the International Space Station (ISS) Water Processor Assembly (WPA) was cultured in a WPA influent water ersatz formulation that is tailored for microbiology studies. A mass transfer rate study was carried out using the ersatz WPA influent water by introducing a water-soluble dye to represent dissolved nutrients and nutrient particles. Imaging of dye diffusion over time allowed for the comparison of mass transport rates under a series of rotation per minute (RPM) speeds for the High Aspect Ratio Vessels (HARVs) on a Rotating Wall Vessel (RWV). This was done to determine the speed that will most accurately simulate the low convective rates experienced under actual µg conditions. Three biological replicates of the Burkholderia contaminans (B. contaminans) microbe were cultured under simulated µg with a rotating (R) control in the horizontal plane at the determined optimal RPM of 15, along with a stationary (S) reference culture. At T=0, and then at T=1,2,3 (in exponential phase) and T=4 (in S phase), the bacterial culture and ersatz were harvested for transcriptomic and nutrient content analysis, respectively. The experimental results illustrated that phosphate is a limiting nutrient in the WPA ersatz formula. Nutrient analysis illustrated that the µg treatment culture took up essential nutrients more rapidly than the R and S control cultures, yet non-essential nutrients remained higher in the µg treatment than in the controls at later timepoints. The rapid uptake and subsequent starvation of phosphate in the culture under µg conditions is further illustrated in the transcriptomic response when compared to that of the R control condition. The subsequent starvation response may serve as one element to explain a moderate enhancement of biofilm formation in the µg treatment. One implication of this work is that biofilm mitigation in the ISS environment could be supported by ensuring a steady flow of water as a vehicle for phosphate within the WPA to avoid complete phosphate consumption, which occurs in times of no flow and leads to undesired biofilm formation.

Aubrie O’Rourke↗

2023 Cleanroom Monitoring for OSIRIS-REx

OSIRIS-REx is an asteroid sample return mission that delivered asteroid regolith from Bennu to Earth on 9/24/2023 [1]. Cleanroom monitoring of OSIRIS-REx Curation facilities has been ongoing since 2021 when construction of the OSIRIS-REx lab at Johnson Space Center (JSC) was completed [2]. The monitoring continues inside of the OSIRIS-REx lab in Houston. Monitoring was also conducted on a temporary cleanroom constructed inside a hangar at the Utah Test and Training Range (UTTR) to initially receive the Sample Return Capsule (SRC) upon its landing there. The following reports on cleanroom monitoring that has taken place in the months leading up to as well as after OSIRIS-REx return. Monitoring techniques used include particle counts, deployment of Si wafer witness plates (Balazs, Inc.) and Al foil witness plates, gas sampling (Balazs, Inc.) and microbiological monitoring. We collected particle counts at both the lab at JSC as well as the temporary cleanroom at UTTR. Particle counts were taken monthly from six locations in the lab at JSC. Particle counts were taken from seven locations at UTTR in July, August, and September. The UTTR cleanroom particle counts were taken daily in the five days leading to OSIRIS-REx return as well as consistently the day of return (every 2 minutes and 15 seconds 7:21 am- 6:27 pm) from one location inside the cleanroom. The particle counts at UTTR consistently stayed well within the ranges of its required ISO 7 equivalent classification (352,000 maximum particles/ m3 ≥ 0.5 μm). Most analysis showed that the particle counts were far below ISO 7 requirements (highest measured count was 61,484 particles/ m3 ≥ 0.5 um). but the elevated instances correlated to exterior hangar doors being open. Particle counts were reduced when keeping exterior hangar doors closed. The particle counts at JSC have stayed consistently within their ISO 5 equivalent classification (3,520 maximum particles/ m3 ≥ 0.5 um). In most instances, the counts are much better except in one instance where the count measured 13,381 particles/ m3 ≥ 0.5 um. The high spike in particle counts in the JSC lab correlated with the air handlers being shut down temporarily the previous day. The particle counts taken in the same location quickly reduced and measured 0 when taken a month later with the air handlers on. We deployed 8-inch semiconductor polished Si wafer witness plates for 24-hour periods inside of the OSIRIS-REx Curation lab at JSC (in July, August, September, and November 2023) and the cleanroom at UTTR (July and September 2023) to test for possible organic and inorganic contaminants. We also deployed these wafers inside of the OSIRIS-REx Touch-and-Go sample Acquisition Mechanism (TAGSAM) glovebox (where Bennu sample processing occurs) and desiccator (Bennu sample storage) at JSC in August 2023. These samples were all sent to Balazs for analysis via Thermal Desorption Gas Chromatography Mass Spectroscopy (TD-GC-MS) to quantify organic compounds and vapor phase decomposition inductively coupled plasma mass spectrometry (VPD-ICP-MS) to quantify inorganic contaminants. We collected air samples using an adsorbent tube connected to a pump for six hours (100 mL/minute) inside of both the JSC lab and the UTTR cleanroom. These samples were taken at JSC in July, August, September, November, and December and at UTTR in July and September. Additionally, a sample was taken just outside of the TAGSAM glovebox airlock door in July. These samples were sent to Balazs for analysis of volatile organics in air. Overall, the OSIRIS-REx lab at JSC and cleanroom at UTTR yielded very low organic and inorganic contaminants (similar results to Genesis lab at JSC which is ISO 4 equivalent) except for boron, which is attributed to the borosilicate glass in the fan filter units. We collected monthly surface and air samples inside the JSC lab to monitor potential microbial contamination. We report the recovery rate for each sampling event, which is defined as the number of samples exhibiting bacterial or fungal growth divided by the total number of samples collected. Since the lab was commissioned in 2021 the median recovery rate is 29%. This recovery rate is consistent with other ISO 5 equivalent labs used to curate astromaterials collections. Increases in recovery rate correspond to construction and/or extra activity in the lab. However, increases in recovery rate do not reliably correlate to increases in particle counts, which highlights the need for a dedicated microbial monitoring program for biologically sensitive collections. We sampled the temporary clean room at UTTR in July, August, and September of 2023 to monitor potential microbial contamination. The recovery rate decreased from 80% in August to 29% in September. However, in all instances, the diversity of bacteria and fungi was higher in the UTTR cleanroom than in the JSC lab. We routinely collected more than twenty different organisms from the UTTR cleanroom. In the JSC lab the median diversity is 2 organisms. Based on these results we hypothesize that microbes from UTTR could be transported back to JSC with the Bennu samples. To mitigate this risk, in September of 2023, we implemented additional cleaning procedures in the JSC lab to reduce the bioburden on surfaces that could come into direct contact with hardware used to process Bennu samples. Since adopting these additional cleaning measures, the median recovery rate has decreased to 14%. We did not observe an increase in fungal or bacterial diversity in the lab in the October sampling. We will continue to monitor this trend closely for the next several months. Supported by NASA under Award NNH09ZDA007O and Contract NNM10AA11C.

Rachel Comstock Funk↗

Ultrashort Pulse Laser Surface Processing Techniques for Sterilization of Metal Surfaces for Planetary Protection

To prevent forward contamination from microbes aboard spacecraft intended to search for extraterrestrial life, there is a need for effective sterilization methods. However, current techniques are both time-consuming and expensive. For example, dry heat sterilization requires removal from the assembly site and several days of treatment. Furthermore, some components such as optics and electronics are not compatible with current sterilization techniques. Here, we report the latest results in our development of a novel femtosecond laser processing technique for the rapid sterilization of spacecraft hardware. Femtosecond lasers produce extremely high photon fluxes (10^29 photons/sec*cm^2, ~0.03 J/cm^2) in extremely short pulses, which can inactivate even stress-tolerant microbial spores with minimal damage to the spacecraft surface. Aluminum coupons were inoculated with specific densities of Bacillus subtilis bacterial endospores. These coupons were treated with various laser illumination parameters. Afterward, metal coupon samples were assayed for viable spores using a polyvinyl alcohol (PVA) peel, serial dilution, and plating for colony-forming units (CFU). Results indicate that with high enough energy density and pulse counts, most bacterial spores are inactivated with minimal damage to the metal. The sterilization is dependent on both the fluence and pulse count. In addition, femtosecond pulses are more effective than longer pulses for inactivation. These experiments have consistently achieved 4-log reduction in viable spores. Sterilization has been achieved on both flat metal coupons and non-flat surfaces with microchannels, with a slight reduction in sterilization efficiency on the uneven surface. The application of air flow during laser processing was also investigated as a way to remove spores that are dislodged from the surface by the laser illumination, which would contribute to the reduction of spacecraft bioburden. With laser processing technology rapidly evolving, our results support the possibility of an extremely rapid, in-situ surface sterilization method for use in spacecraft assembly clean rooms.

Kaleb McQuillan↗

Does the International Space Station Leak DNA? Preliminary Results from the ISS External Microorganisms Payload

Existing crewed spacecraft like the ISS (International Space Station) leak by design. The ISS routinely releases gas to maintain life support systems and when astronauts exit the station to perform space walks. The chemical component of this leakage is well characterized, but the biological components are not. The ISS is not subject to planetary protection requirements, but planned missions to Mars will use similar systems and will be subject to planetary protection requirements. If detectable microorganisms are escaping through vents and or airlocks we may need to redesign our crewed habitats to minimize this type of contamination. To test the hypothesis that microorganisms from inside ISS are detectable on exterior surfaces an astronaut used the ISS External Microorganisms sampling kit (Rucker et al. 2018) to sample exterior surfaces of the ISS during an EVA (Extra Vehicular Activity) in January of 2025. These samples were returned to Earth for DNA extraction and sequencing. We successfully, extracted and sequenced bacterial, fungal and viral DNA from these samples that was not present in the negative controls. These results should help NASA refine the planetary protection requirements for crewed missions. Methods: The samples were collected using sterile, DNA free, buccal swabs (23 mm. diameter) housed in custom canisters. Each canister uses a 0.2 μm Teflon filter to maintain sterility as the caddy, holding 8 swabs moves in and out of vacuum. The astronaut sampled the: 1) airlock vestibule, 2) airlock thermal cover, 3) a gap in the micrometeorite shielding near the airlock, 4) a handrail near the airlock, 5) the Carbon Dioxide Removal Assembly vent, and 6) the Vacuum Exhaust System vent. The seventh swab was exposed to vacuum during the EVA without touching it to a surface. The eighth swab, a negative control, was not opened until the caddy returned to Earth. DNA was extracted from the swabs using a QIamp UCP Pathogen kit and prepared for sequencing on an Aviti (Element Biosciences) sequencer (Arslan et al. 2024). The resulting sequences were analyzed using the EDGE Bioinformatics platform (Li et al. 2017). The sequences were analyzed individually using tools like BLAST, GOTTCHA2, Kraken2, and PanGIA. The data were also assembled into metagenome assembled genomes) using tools like CONCOCT, MaxBin2 and MetaBAT2. Results: We successfully extracted and sequenced bacterial, archaeal, fungal and viral DNA from all seven samples. The handrail swab had the lowest number of reads (768,651) and the airlock thermal cover had the highest number of reads (8,819,230). These samples contain DNA from human associated bacteria (e.g. Crynebacterium riegelii ), fungi (.e.g. Penicillium rubens ), and viruses (e.g Alphapapillomavirus ). Conclusion: Preliminary interpretation suggest that the airlock and the space suits themselves are the largest sources of contaminant DNA. Most if not all of the DNA is from organisms known to be present inside the ISS. Vents attached to life support systems may be a lesser source of biological contamination. Further analysis should help NASA address planetary protection knowledge gaps for crewed missions.

Aaron B Regberg↗

Low-Shear modeled microgravity alters the Salmonella enterica serovar typhimurium stress response in an RpoS-independent manner

We have previously demonstrated that low-shear modeled microgravity (low-shear MMG) serves to enhance the virulence of a bacterial pathogen, Salmonella enterica serovar Typhimurium. The Salmonella response to low-shear MMG involves a signaling pathway that we have termed the low-shear MMG stimulon, though the identities of the low-shear MMG stimulon genes and regulatory factors are not known. RpoS is the primary sigma factor required for the expression of genes that are induced upon exposure to different environmental-stress signals and is essential for virulence in mice. Since low-shear MMG induces a Salmonella acid stress response and enhances Salmonella virulence, we reasoned that RpoS would be a likely regulator of the Salmonella low-shear MMG response. Our results demonstrate that low-shear MMG provides cross-resistance to several environmental stresses in both wild-type and isogenic rpoS mutant strains. Growth under low-shear MMG decreased the generation time of both strains in minimal medium and increased the ability of both strains to survive in J774 macrophages. Using DNA microarray analysis, we found no evidence of induction of the RpoS regulon by low-shear MMG but did find that other genes were altered in expression under these conditions in both the wild-type and rpoS mutant strains. Our results indicate that, under the conditions of these studies, RpoS is not required for transmission of the signal that induces the low-shear MMG stimulon. Moreover, our studies also indicate that low-shear MMG can be added to a short list of growth conditions that can serve to preadapt an rpoS mutant for resistance to multiple environmental stresses.

Non-NASA Center↗

Microarray analysis identifies Salmonella genes belonging to the low-shear modeled microgravity regulon

The low-shear environment of optimized rotation suspension culture allows both eukaryotic and prokaryotic cells to assume physiologically relevant phenotypes that have led to significant advances in fundamental investigations of medical and biological importance. This culture environment has also been used to model microgravity for ground-based studies regarding the impact of space flight on eukaryotic and prokaryotic physiology. We have previously demonstrated that low-shear modeled microgravity (LSMMG) under optimized rotation suspension culture is a novel environmental signal that regulates the virulence, stress resistance, and protein expression levels of Salmonella enterica serovar Typhimurium. However, the mechanisms used by the cells of any species, including Salmonella, to sense and respond to LSMMG and identities of the genes involved are unknown. In this study, we used DNA microarrays to elucidate the global transcriptional response of Salmonella to LSMMG. When compared with identical growth conditions under normal gravity (1 x g), LSMMG differentially regulated the expression of 163 genes distributed throughout the chromosome, representing functionally diverse groups including transcriptional regulators, virulence factors, lipopolysaccharide biosynthetic enzymes, iron-utilization enzymes, and proteins of unknown function. Many of the LSMMG-regulated genes were organized in clusters or operons. The microarray results were further validated by RT-PCR and phenotypic analyses, and they indicate that the ferric uptake regulator is involved in the LSMMG response. The results provide important insight about the Salmonella LSMMG response and could provide clues for the functioning of known Salmonella virulence systems or the identification of uncharacterized bacterial virulence strategies.

Non-NASA Center↗

Rationally designed fluorescently labeled sulfate-binding protein mutants: evaluation in the development of a sensing system for sulfate

Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the development of reagentless sensing systems for their corresponding ligands. In this work, we evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe and rationally designed a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. An environment-sensitive fluorescent probe (MDCC) was then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. Three different environment-sensitive fluorescent probes (1,5-IAEDANS, MDCC, and acylodan) were studied with the SBP171 mutant protein. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the submicromolar range was developed. Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 517-526, 2002.

Evaluation Studies↗

Spatial distribution of total, ammonia-oxidizing, and denitrifying bacteria in biological wastewater treatment reactors for bioregenerative life support

Bioregenerative life support systems may be necessary for long-term space missions due to the high cost of lifting supplies and equipment into orbit. In this study, we investigated two biological wastewater treatment reactors designed to recover potable water for a spacefaring crew being tested at Johnson Space Center. The experiment (Lunar-Mars Life Support Test Project-Phase III) consisted of four crew members confined in a test chamber for 91 days. In order to recycle all water during the experiment, an immobilized cell bioreactor (ICB) was employed for organic carbon removal and a trickling filter bioreactor (TFB) was utilized for ammonia removal, followed by physical-chemical treatment. In this study, the spatial distribution of various microorganisms within each bioreactor was analyzed by using biofilm samples taken from four locations in the ICB and three locations in the TFB. Three target genes were used for characterization of bacteria: the 16S rRNA gene for the total bacterial community, the ammonia monooxygenase (amoA) gene for ammonia-oxidizing bacteria, and the nitrous oxide reductase (nosZ) gene for denitrifying bacteria. A combination of terminal restriction fragment length polymorphism (T-RFLP), sequence, and phylogenetic analyses indicated that the microbial community composition in the ICB and the TFB consisted mainly of Proteobacteria, low-G+C gram-positive bacteria, and a Cytophaga-Flexibacter-Bacteroides group. Fifty-seven novel 16S rRNA genes, 8 novel amoA genes, and 12 new nosZ genes were identified in this study. Temporal shifts in the species composition of total bacteria in both the ICB and the TFB and ammonia-oxidizing and denitrifying bacteria in the TFB were also detected when the biofilms were compared with the inocula after 91 days. This result suggests that specific microbial populations were either brought in by the crew or enriched in the reactors during the course of operation.

NASA Discipline Life Support Systems↗

Natural transfer of viable microbes in space

The possibility and probability of natural transfer of viable microbes from Mars to Earth and Earth to Mars traveling in meteoroids during the first 0.5 Ga and the following 4 Ga are investigated, including: --radiation protection against the galactic cosmic ray nuclei and the solar rays, dose rates as a function of the meteorite's radial column mass (radius x density), combined with dose rates generated by natural radioactivity within the meteorite; and survival curves for some bacterial species using NASA's HZETRN transport code --other factors affecting microbe survival: vacuum; central meteorite temperatures at launch, orbiting, and arrival; pressure and acceleration at launch; spontaneous DNA decay; metal ion migration --mean sizes and numbers of unshocked meteorites ejected and percentage falling on Earth, using current semiempirical results --viable flight times for the microbe species Bacillus subtilis and Deinococcus radiodurans R1 --the approximate fraction of microbes (with properties like the two species studied) viably arriving on Earth out of those ejected from Mars during the period 4 Ga BP to the present time, and during the 700 Ma from 4.5 to 3.8 Ga. Similarly, from Earth to Mars. The conclusion is that if microbes existed or exist on Mars, viable transfer to Earth is not only possible but also highly probable, due to microbes' impressive resistance to the dangers of space transfer and to the dense traffic of billions of martian meteorites which have fallen on Earth since the dawn of our planetary system. Earth-to-Mars transfer is also possible but at a much lower frequency.

Non-NASA Center↗

Association of a new type of gliding, filamentous, purple phototrophic bacterium inside bundles of Microcoleus chthonoplastes in hypersaline cyanobacterial mats

An unidentified filamentous purple bacterium, probably belonging to a new genus or even a new family, is found in close association with the filamentous, mat-forming cyanobacterium Microcoleus chthonoplastes in a hypersaline pond at Guerrero Negro, Baja California Sur, Mexico, and in Solar Lake, Sinai, Egypt. This organism is a gliding, segmented trichome, 0.8-0.9 micrometer wide. It contains intracytoplasmic stacked lamellae which are perpendicular and obliquely oriented to the cell wall, similar to those described for the purple sulfur bacteria Ectothiorhodospira. These bacteria are found inside the cyanobacterial bundle, enclosed by the cyanobacterial sheath. Detailed transmission electron microscopical analyses carried out in horizontal sections of the upper 1.5 mm of the cyanobacterial mat show this cyanobacterial-purple bacterial association at depths of 300-1200 micrometers, corresponding to the zone below that of maximal oxygenic photosynthesis. Sharp gradients of oxygen and sulfide are established during the day at this microzone in the two cyanobacterial mats studied. The close association, the distribution pattern of this association and preliminary physiological experiments suggest a co-metabolism of sulfur by the two-membered community. This probable new genus of purple bacteria may also grow photoheterotrophically using organic carbon excreted by the cyanobacterium. Since the chemical gradients in the entire photic zone fluctuate widely in a diurnal cycle, both types of metabolism probably take place. During the morning and afternoon, sulfide migrates up to the photic zone allowing photoautotrophic metabolism with sulfide as the electron donor. During the day the photic zone is highly oxygenated and the purple bacteria may either use oxidized species of sulfur such as elemental sulfur and thiosulfate in the photoautotrophic mode or grow photoheterotrophically using organic carbon excreted by M. chthonoplastes. The new type of filamentous purple sulfur bacteria is not available yet in pure culture, and its taxonomical position cannot be fully established. This organism is suggested to be a new type of gliding, filamentous, purple phototroph.

NASA Discipline Exobiology↗

Phylogenetic diversity and position of the genus Campylobacter

RNA sequence analysis has been used to examine the phylogenetic position and structure of the genus Campylobacter. A complete 5S rRNA sequence was determined for two strains of Campylobacter jejuni and extensive partial sequences of the 16S rRNA were obtained for several strains of C. jejuni and Wolinella succinogenes. In addition limited partial sequence data were obtained from the 16S rRNAs of isolates of C. coli, C. laridis, C. fetus, C. fecalis, and C. pyloridis. It was found that W. succinogenes is specifically related to, but not included, in the genus Campylobacter as presently constituted. Within the genus significant diversity was noted. C. jejuni, C. coli and C. laridis are very closely related but the other species are distinctly different from one another. C. pyloridis is without question the most divergent of the Campylobacter isolates examined here and is sufficiently distinct to warrant inclusion in a separate genus. In terms of overall position in bacterial phylogeny, the Campylobacter/Wolinella cluster represents a deep branching most probably located within an expanded version of the Division containing the purple photosynthetic bacteria and their relatives. The Campylobacter/Wolinella cluster is not specifically includable in either the alpha, beta or gamma subdivisions of the purple bacteria.

NASA Discipline Exobiology↗

Removal of Burkholderia cepacia biofilms with oxidants

Iodine is used to disinfect the water system aboard US space shuttles and is the anticipated biocide for the international space station. Water quality on spacecraft must be maintained at the highest possible levels for the safety of the crew. Furthermore, the treatment process used to maintain the quality of water on research must be robust and operate for long periods with minimal crew intervention. Biofilms are recalcitrant and pose a major threat with regard to chronic contamination of spacecraft water systems. We measured the effectiveness of oxidizing biocides on the removal and regrowth of Burkholderia (Pseudomonas) cepacia biofilms. B. cepacia, isolated from the water distribution system of the space shuttle Discovery, was grown in continuous culture to produce a bacterial contamination source for biofilm formation and removal studies. A 10(7) CFU ml-1 B. cepacia suspension, in distilled water, was used to form biofilms on 3000 micrometers2 glass surfaces. Rates of attachment were measured directly with image analysis and were found to be 7.8, 15.2, and 22.8 attachment events h-1 for flow rates of 20.7, 15.2, and 9.8 ml min-1, respectively. After 18 h of formation, the B. cepacia biofilms were challenged with oxidants (ozone, chlorine, and iodine) and the rates of biofilm removal determined by image analysis. Fifty percent of the biofilm material was removed in the first hour of continous treatment with 24 mg l-1 chlorine or 2 mg l-1 ozone. Iodine (48 mg l-1) did not remove any measurable cellular material after 6 h continuous contact. After this first removal of biofilms by the oxidants, the surface was allowed to refoul and was again treated with the biocide. Iodine was the only compound that was unable to remove cellular debris from either primary or secondary biofilms. Moreover, treating primary biofilms with iodine increased the rate of formation of secondary biofilms, from 4.4 to 5.8 attachment events h-1. All the oxidants tested inactivated the B. cepacia associated with both primary and secondary biofilms. The amount of biocide needed to inactivate 50% of planktonic B. cepacia in 10 min at 25 degrees C was 8.4, 0.5, and 0.2 mg l-1 for iodine, chlorine, and ozone, respectively. The data suggest that iodine maynot be the best chemical for treating of biofilms when removal of cellular material is required.

NASA Discipline Environmental Health↗

On the use of antibiotics to reduce rhizoplane microbial populations in root physiology and ecology investigations

No straightforward method exists for separating the proportion of ion exchange and respiration due to rhizoplane microbial organisms from that of root ion exchange and respiration. We examined several antibiotics that might be used for the temporary elimination of rhizoplane bacteria from hydroponically grown wheat roots (Triticum aestivum cv. Veery 10). Each antibiotic was tested for herbicidal activity and plate counts were used to enumerate bacteria and evaluate antibiotic kinetics. Only lactam antibiotics (penicillins and cephalosporins) did not reduce wheat growth rates. Aminoglycosides, the pyrimidine trimethoprim, colistin and rifampicin reduced growth rates substantially. Antibiotics acted slowly, with maximum reductions in rhizoplane bacteria occurring after more than 48 h of exposure. Combinations of nonphytotoxic antibiotics reduced platable rhizoplane bacteria by as much as 98%; however, this was generally a reduction from about 10(9) to 10(6) colony forming units per gram of dry root mass, so that many viable bacteria remained on root surfaces. We present evidence which suggests that insufficient bacterial biomass exists on root surfaces of nonstressed plants grown under well-aerated conditions to quantitatively interfere with root nitrogen absorption measurements.

NASA Discipline Life Support Systems↗

Diverse Thermus species inhabit a single hot spring microbial mat

Through an effort to characterize aerobic chemoorganotrophic bacteria in the Octopus Spring cyano-bacterial mat community, we cultivated four Thermus isolates with unique 16S rRNA sequences. Isolates clustered within existing Thermus clades, including those containing Thermus ruber, Thermus aquaticus, and a subgroup closely related to T. aquaticus. One Octopus Spring isolate is nearly identical (99.9% similar) to isolates from Iceland, and two others are closely related to a T. ruber isolated from Russia. Octopus Spring isolates similar to T. aquaticus and T. ruber exhibited optimal growth rates at high (65-70 degrees C) and low (50 degrees C) temperatures, respectively, with the most abundant species best adapted to the temperature of the habitat (50-55 degrees C). Our results display a diversity of Thermus genotypes defined by 16S rRNA within one hot spring microbial community. We suggest that specialization to temperature and perhaps other local environmental features controls the abundance of Thermus populations.

Non-NASA Center↗

Tubulinlike protein from Spirochaeta bajacaliforniensis

Tubulin proteins are the fundamental subunits of all polymeric microtubule-based eukaryotic structures. Long, hollow structures each composed of 13 protofilaments as revealed by electron microscopy, microtubules (240 angstroms in diameter) are nearly ubiquitous in eukaryotes. These proteins have been the subject of intense biochemical and biophyiscal interest since the early 1970s and are of evolutionary interest as well. If tubulin-based structures (i.e., neurotubules, mitotic spindle tubules, centrioles, kinetosomes, axonemes, etc.) evolved from spirochetes by way of motility symbioses, tubulin homologies with spirochete proteins should be detectable. Tubulin proteins are widely thought to be limited to eukaryotes. Yet both azotobacters and spirochetes have shown immunological cross-reactivity with antitubulin antibodies. In neither of these studies was tubulin isolated nor any specific antigen identified as responsible for the immunoreactivity. Furthermore, although far less uniform in structure than eukaryotic microtubules, various cytoplasmic fibers and tubules (as seen by electron microscopy) have been reported in several types of prokaryotes (e.g., Spirochaeta; large termite spirochetes; treponemes; cyanobacteria; and Azotobacter. This work forms a part of our long-range study of the possible prokaryotic origin of tubulin and microtubules. Spirochetes are helically shaped gram-negative motile prokaryotes. They differ from all other bacterial in that the position of their flagella is periplasmic: their flagella lie between the inner and outer membranes of the gram-negative cell wall. Some of the largest spirochetes have longitudinally aligned 240 angstrom microtubules. Unfortunately, in spite of many attempts, all of the larger spirochetes (family Pillotaceae) with well-defined cytoplasmic tubules and antitubulin immunoreactivity are not cultivable. However, a newly described spirochete species (Spirochaeta bajacaliforniensis) possessing cytoplasmic fibers displays antitubulin immunoreactivity in whole-cell preparations. Since preliminary observations suggested that Spirochaeta bajacaliforniensis proteins may be related to eukaryotic tubulins, their characterization was undertaken. Brain tubulin can be purified by utilizing its ability to polymerize at warm temperatures and to depolymerize in the cold. After several cycles of sedimentation and redissolution the microtubule fraction is comprised of 75% tubulin and 20% high molecular mass microtubule-associated proteins (MAPs). In this paper we report that components of cell lysates, prepared from a spirochete that contains cytoplasmic fibers (Spirochaeta bajacaliforniensis), also exhibit the property of temperature-dependent cyclical sedimentation. Additionally we report the identification and characterization of the polypeptide responsible for cross-reactivity with antitubulin antiserum.

Non-NASA Center↗

Identification of a small tetraheme cytochrome c and a flavocytochrome c as two of the principal soluble cytochromes c in Shewanella oneidensis strain MR1

Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c(3) but define a new folding motif for small multiheme cytochromes c.

Cytochrome c Group/chemistry/genetics/metabolism↗