Evaluating the biological effectiveness of space flight factors by means of the lysogenic bacteria e. coli k-12 /lambda/
Biological experiments with lysogenic bacteria to determine genetic effects of space flight factors
BACTERIA↗
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A RELATIONSHIP BETWEEN GROWTH AND GRAVITY IN BACTERIA
Experimental investigation of the effect of gravity on the growth of broth cultures of e. coli bacteria
BACTERIOLOGY↗
Direct Isolation of Purines and Pyrimidines from Nucleic Acids Using Sublimation
A sublimation technique was developed to isolate purines and pyrimidines directly from lambda-deoxyribonucleic acid (lambda-DNA) and Escherichia coli cells. The sublimation of adenine, cytosine, guanine, and thymine from lambda-DNA was tested under reduced pressure (approx. 0.5 Torr) at temperatures of >150 C. With the exception of guanine, approximately 60 -75% of each base was sublimed directly from the lambda-DNA and recovered on a coldfinger of the sublimation apparatus after heating to 450 C. Several nucleobases including adenine, cytosine, thymine, and uracil were also recovered from E. coli bacteria after heating the cells to the same temperature, although some thermal decomposition of the bases also occurred. These results demonstrate the feasibility of using sublimation to isolate purines and pyrimidines from native E. coli DNA and RNA without any chemical treatment of the cells.
Glavin, Daniel P.↗
Environmental Control Of A Genetic Process
E. coli bacteria altered to contain DNA sequence encoding production of hemoglobin made to produce hemoglobin at rates decreasing with increases in concentration of oxygen in culture media. Represents amplification of part of method described in "Cloned Hemoglobin Genes Enhance Growth Of Cells" (NPO-17517). Manipulation of promoter/regulator DNA sequences opens promising new subfield of recombinant-DNA technology for environmental control of expression of selected DNA sequences. New recombinant-DNA fusion gene products, expression vectors, and nucleotide-base sequences will emerge. Likely applications include such aerobic processes as manufacture of cloned proteins and synthesis of metabolites, production of chemicals by fermentation, enzymatic degradation, treatment of wastes, brewing, and variety of oxidative chemical reactions.
Khosla, Chaitan↗
On-Orbit Quantitative Real-Time Gene Expression Analysis Using the Wetlab-2 System
NASA Ames Research Center's WetLab-2 Project enables on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The resulting RNA is dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The project selected the Cepheid SmartCycler (TradeMark), a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, because of its advantages including rugged modular design, low power consumption, rapid thermal ramp times and four-color multiplex detection. Single tube multiplex assays can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to conduct qRT-PCR and generate results on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also eliminate the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples and provide on-orbit gene expression benchmarking prior to sample return. Finally, the system can also be used for analysis of air, surface, water, and clinical samples to monitor environmental pathogens and crew health. The validation flight of the WetLab-2 system using E. coli bacteria and mouse liver launched on SpaceX-7 in June 2015 and will remain on the ISS National Laboratory.
ISS tools↗
On-orbit RNA Purification and qRT-PCR Capabilities of the WetLab-2 System
NASA Ames Research Center’s WetLab-2 system brings new capabilities to the ISS for researchers. The system can lyse cells and extract RNA on-orbit from different sample types ranging from microbial cultures to animal tissues. Our purification method has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The purified RNA can then either be stabilized for return to Earth or can be used to conduct on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. qRT-PCR reactions are performed by dispensing the RNA into reaction tubes that contain all lyophilized reagents needed to perform the analysis. The system uses a Cepheid® SmartCycler that allows for multiplexing of assays, this can be used to normalize for RNA concentration and integrity and to study multiple genes of interest in each tube. There are a total of 16 independent PCR modules each capable of detecting up to four fluorescent channels. The WetLab- 2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to purify and stabilize RNA on-orbit can eliminate the confounding effects of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of some samples. It also has the benefit of minimizing the needed downmass. Conducting qRT-PCR and generating results on-orbit is also an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also provide benchmarking prior to sample return. The system can also be used for analysis of air, surface, water, and clinical samples to monitor environmental pathogens and crew health. The validation flight of the WetLab-2 system using E. coli bacteria and mouse liver is scheduled to launch on SpaceX-8 this spring. Pending operations, the preliminary results from the validation flight will be presented. To support the needs of future researchers, we are adapting our system to purify RNA from two additional sample types: fibrous tissue such as muscle and mammalian adherent cells grown on alginate beads. Progress of this work will also be presented. The WetLab-2 Project is supported by the Research Integration Office in the ISS Program.
Parra, Macarena↗
The Antibio experiment
An experiment was flown on Spacelab to confirm the results of the Cytos 2 experiment on Salyut 7, which found an increase in minimal inhibitory concentration in in-flight cultures, i.e., an increase of antibiotic resistance. The 1 g centrifuge on Biorack was also used to differentiate the effects of cosmic rays and microgravity. The antibiotic sensitivity of bacteria cultivated in vitro during orbital flight was studied. The bacteria was E. coli, the antibiotic was Colistin. An increase of antibiotic resistance is observed. Three explanations are offered: stimulation of bacterial proliferation in space; a relationship between the transport of antibiotics into cells and modifications of cellular envelope permeability; and a combined effect of both phenomena.
Lapchine, L.↗
Evaluation of Low-Pressure Cold Plasma for Disinfection for ISS Grown Produce and Metallic Instrumentation
Cold plasma (CP) cleaning is a dry, non-thermal process, which can provide broad-spectrum antimicrobial activity yet reportedly causes little to no damage to the object being sanitized. Since cold plasma uses no liquids, it has the distinct advantage when used in microgravity of not having to separate liquids from the item being cleaned. This paper will present results on an effort to use low pressure CP to disinfect or sterilize materials for in space applications. Exposure times from 0 to 60 minutes and pressures ranging from 10 to 100 Pa were used to optimize plasma parameters to achieve acceptable kill rates for 3 bacteria, Bacillus cereus, E. coli and Bacillus pumulis SAFR-32 and one fungi, Aspergillus niger. These tests were done on produce and metal coupons to simulate medical equipment. Produce testing was not successful, with unacceptable kill rates and the produce being negatively impacted by exposure to the plasma. The plasma caused a 5 log reduction in the number of viable bacteria on metal coupon tests, which placed the number of viable bacteria below the detection limit. This is a very promising result showing that sterilization of medical equipment with cold plasma is feasible. Scanning Electron Microscope images were taken before and after exposure. The images after plasma exposure shows that the bacteria spores have been physically affected, as their size has gotten smaller and the appearance has changed.
Hintze, Paul E.↗
Differential effects of catecholamines on in vitro growth of pathogenic bacteria
Supplementation of minimal medium inoculated with bacterial cultures with norepinephrine, epinephrine, dopamine, or isoproterenol resulted in marked increases in growth compared to controls. Norepinephrine and dopamine had the greatest enhancing effects on growth of cultures of Pseudomonas aeruginosa and Klebsiella pneumoniae, while epinephrine and isoproterenol also enhanced growth to a lesser extent. The growth of Escherichia coli in the presence of norepinephrine was greater than growth in the presence of the three other neurochemicals used in the study. Growth of Staphylococcus aureus was also enhanced in the presence of norepinephrine, but not to the same degree as was the growth of gram negative bacteria. Addition of culture supernatants from E. coli cultures that had been grown in the presence of norepinephrine was able to enhance the growth of K. pneumoniae. Addition of the culture supernatant fluid culture from E. coli cultures that had been grown in the presence of norepinephrine did not enhance growth of P. aeruginosa or S. aureus. Culture supernatant fluids from bacteria other than E. coli grown in the presence of norepinephrine were not able to enhance the growth of any bacteria tested. The results suggest that catecholamines can enhance growth of pathogenic bacteria, which may contribute to development of pathogenesis; however, there is no uniform effect of catecholamines on bacterial growth.
Non-NASA Center↗
Microbiological methods for the water recovery systems test, revision 1.1
Current microbiological parameters specified to verify microbiological quality of Space Station Freedom water quality include the enumeration of total bacteria, anaerobes, aerobes, yeasts and molds, enteric bacteria, gram positives, gram negatives, and E. coli. In addition, other parameters have been identified as necessary to support the Water Recovery Test activities to be conducted at the NASA/MSFC later this year. These other parameters include aerotolerant eutrophic mesophiles, legionellae, and an additional method for heterotrophic bacteria. If inter-laboratory data are to be compared to evaluate quality, analytical methods must be eliminated as a variable. Therefore, each participating laboratory must utilize the same analytical methods and procedures. Without this standardization, data can be neither compared nor validated between laboratories. Multiple laboratory participation represents a conservative approach to insure quality and completeness of data. Invariably, sample loss will occur in transport and analyses. Natural variance is a reality on any test of this magnitude and is further enhanced because biological entities, capable of growth and death, are specific parameters of interest. The large variation due to the participation of human test subjects has been noted with previous testing. The resultant data might be dismissed as 'out of control' unless intra-laboratory control is included as part of the method or if participating laboratories are not available for verification. The purpose of this document is to provide standardized laboratory procedures for the enumeration of certain microorganisms in water and wastewater specific to the water recovery systems test. The document consists of ten separate cultural methods and one direct count procedure. It is not intended nor is it implied to be a complete microbiological methods manual.
Rhoads, Tim↗
Sensitive detection of Escherichia coli O157:H7 in food and water by immunomagnetic separation and solid-phase laser cytometry
Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r2 = 0. 95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r2 = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.
Non-NASA Center↗
Gentamicin: effect on E. coli in space
Previous investigations have shown that liquid bacterial cultures grown in space flight were not killed as effectively by antibiotic treatments as were cultures grown on Earth. However, the cause for the decreased antibiotic effectiveness remains unknown. Possible explanations include modified cell proliferation and modified antibiotic transport in the culture medium. Escherichia coli cultures were grown in space flight (STS-69 and STS-73), with and without gentamicin, on a solid agar substrate thus eliminating fluid effects and reducing the unknowns associated with space-flight bacterial cultures in suspension. This research showed that E. coli cultures grown in flight on agar for 24 to 27 hours experienced a heightened growth compared to simultaneous controls. However, addition of gentamicin to the agar killed the bacteria such that both flight and ground control E. coli samples had similar final cell concentrations. Therefore, while the reported existence of a decrease in antibiotic effectiveness in liquid cultures remains unexplained, these data suggest that gentamicin in space flight was at least as effective as, if not more effective than, on Earth, when E. coli cells were grown on agar.
STS-69 Shuttle Project↗
Maintaining the Body's Immune System: Incidence of Latent Virus Shedding During Space Flight
Your body protects you from illness with its own security system - the immune system. This system keeps illness at bay not only by mounting a defense against foreign organisms, but also by controlling the population of bacteria and viruses that normally live in your body. But there's no need to panic: certain microbes can actually exist in your body without causing illness. Some bacteria are even beneficial - like the E. coli in the large intestine that are an important source of vitamin K. While viruses are not exactly considered beneficial, they can also inhabit the human body without causing immediate harm or infection. A good example is the herpes simplex virus type 1 (HSV1), more commonly known as cold sores or fever blisters. This virus infects 70 to 80 percent of all adults but remains latent much of the time. While latent, the virus within cells remains dormant. Activation of the dormant virus causes it to make copies of itself (known as replication) constantly detectable in body fluids such as urine or saliva in a process called shedding. When a person becomes sick or stressed, however, this weakened condition allows the virus to reactivate and multiply. These elevated levels may be enough to produce symptoms, but shedding can also occur without symptoms. This ability to shed without showing signs of infection, or asymptomatic shedding, is of great interest, as it increases the chances of infecting others. The stresses associated with space flight - adapting to microgravity, isolation from family and friends, living and working in a confined space, sleep deprivation, and busy schedules, to name but a few - may weaken astronauts' immune systems, leaving them at greater risk of viral reactivation. Members of the STS-107 crew will participate in this experiment, Incidence of Latent Viral Shedding in Space Flight, to help scientists understand how reactivation works in space, and at what level replication reaches before symptoms begin to show. This study also promises more insight into the behavior of the larger virus family, herpesvirus, which will help us understand how to prevent infection in populations on Earth and reactivation in those already infected.
Pierson, Duane↗
The nucleotide sequence of Beneckea harveyi 5S rRNA
The primary sequence of the 5S ribosomal RNA isolated from the free-living bioluminescent marine bacterium Beneckea harveyi is reported and discussed in regard to indications of phylogenetic relationships with the bacteria Escherichia coli and Photobacterium phosphoreum. Sequences were determined for oligonucleotide products generated by digestion with ribonuclease T1, pancreatic ribonuclease and ribonuclease T2. The presence of heterogeneity is indicated for two sites. The B. harveyi sequence can be arranged into the same four helix secondary structures as E. coli and other prokaryotic 5S rRNAs. Examination of the 5S-RNS sequences of the three bacteria indicates that B. harveyi and P. phosphoreum are specifically related and share a common ancestor which diverged from an ancestor of E. coli at a somewhat earlier time, consistent with previous studies.
Luehrsen, K. R.↗
Several methods for concentrating bacteria in fluid samples
The sensitivities of the firefly luciferase - ATP flow system and luminol flow system were established as 300,000 E. coli per milliliter and 10,000 E. coli per milliliter respectively. To achieve the detection limit of 1,000 bacteria per milliliter previously established, a method of concentrating microorganisms using a sartorius membrane filter system is investigated. Catalase in 50% ethanol is found to be a stable luminol standard and can be used up to 24 hours with only a 10% loss of activity. The luminol reagent is also stable over a 24 hour period. A method of preparing relatively inexpensive luciferase from desiccated firefly tails is developed.
Thomas, R. R.↗
Methods for generation of reporter phages and immobilization of active bacteriophages on a polymer surface
Novel reporter bacteriophages are provided. Provided are compositions and methods that allow bacteriophages that are used for specific detection or killing of E. coli 0157:H7 to be propagated in nonpathogenic E. coli, thereby eliminating the safety and security risks of propagation in E. coli 0157:H7. Provided are compositions and methods for attaching active bacteriophages to the surface of a polymer in order to kill target bacteria with which the phage comes into contact. Provided are modified bacteriophages immobilized to a surface, which capture E. coli 0157:H7 and cause the captured cells to emit light or fluorescence, allowing detection of the bacteria in a sample.
Applegate, Bruce Michael↗
Innate Immune Responses of Drosophila Melanogaster are Altered by Spaceflight
Alterations and impairment of immune responses in humans present a health risk for space exploration missions. The molecular mechanisms under pinning innate immune defense can be confounded by the complexity of the acquired immune system of humans. Drosophila (fruit fly) innate immunity is simpler, and shares many similarities with human innate immunity at the level of molecular and genetic pathways. The goals of this study were to elucidate fundamental immune processes in Drosophila affected by spaceflight and to measure host-pathogen responses post-flight. Five containers, each containing ten female and five male fruit flies, were housed and bred on the space shuttle (average orbit altitude of330.35 km) for 12 days and 18.5 hours. A new generation of flies was reared in microgravity. In larvae, the immune system was examined by analyzing plasmatocyte number and activity in culture. In adults, the induced immune responses were analyzed by bacterial clearance and quantitative real-time polymerase chain reaction (qPCR) of selected genes following infection with E. coli. The RNA levels of relevant immune pathway genes were determined in both larvae and adults by microarray analysis. The ability of larval plasmatocytes to phagocytose E. coli in culture was attenuated following spaceflight, and in parallel, the expression of genes involved in cell maturation was down regulated. In addition, the level of constitutive expression of pattern recognition receptors and opsonins that specifically recognize bacteria, and of lysozymes, antimicrobial peptide (AMP) pathway and immune stress genes, hallmarks of humoral immunity, were also reduced in larvae. In adults, the efficiency of bacterial clearance measured in vivo following a systemic infection with E. coli post-flight, remained robust. We show that spaceflight altered both cellular and humoral immune responses in Drosophila and that the disruption occurs at multiple interacting pathways.
Marcu, Oana↗
Population dynamics of transgenic strain Escherichia coli Z905/pPHL7 in freshwater and saline lake water microcosms with differing microbial community structures
Populations of Escherichia coli Z905/pPHL7, a transgenic microorganism, were heterogenic in the expression of plasmid genes when adapting to the conditions of water microcosms of various mineralization levels and structure of microbial community. This TM has formed two subpopulations (ampicillin-resistant and ampicillin-sensitive) in every microcosm. Irrespective of mineralization level of a microcosm, when E. coli Z905/pPHL7 alone was introduced, the ampicillin-resistant subpopulation prevailed, while introduction of the TM together with indigenous bacteria led to the dominance of the ampicillin-sensitive subpopulation. A high level of lux gene expression maintained longer in the freshwater microcosms than in sterile saline lake water microcosms. A horizontal gene transfer has been revealed between the jointly introduced TM and Micrococcus sp. 9/pSH1 in microcosms with the Lake Shira sterile water. c2005 COSPAR. Published by Elsevier Ltd. All rights reserved.
Ecosystem↗