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1.  PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip ▿ † ‡  
Applied and Environmental Microbiology  2011;77(18):6313-6322.
Environmental microbial community analysis typically involves amplification by PCR, despite well-documented biases. We have developed two methods of PCR-independent microbial community analysis using the high-density microarray PhyloChip: direct hybridization of 16S rRNA (dirRNA) or rRNA converted to double-stranded cDNA (dscDNA). We compared dirRNA and dscDNA communities to PCR-amplified DNA communities using a mock community of eight taxa, as well as experiments derived from three environmental sample types: chromium-contaminated aquifer groundwater, tropical forest soil, and secondary sewage in seawater. Community profiles by both direct hybridization methods showed differences that were expected based on accompanying data but that were missing in PCR-amplified communities. Taxon richness decreased in RNA compared to that in DNA communities, suggesting a subset of 20% in soil and 60% in groundwater that is active; secondary sewage showed no difference between active and inactive populations. Direct hybridization of dscDNA and RNA is thus a viable alternative to PCR-amplified microbial community analysis, providing identification of the active populations within microbial communities that attenuate pollutants, drive global biogeochemical cycles, or proliferate disease states.
PMCID: PMC3187179  PMID: 21764955
2.  Diversity of Anaerobic Microbes in Spacecraft Assembly Clean Rooms ▿ †  
Although the cultivable and noncultivable microbial diversity of spacecraft assembly clean rooms has been previously documented using conventional and state-of-the-art molecular techniques, the occurrence of obligate anaerobes within these clean rooms is still uncertain. Therefore, anaerobic bacterial communities of three clean-room facilities were analyzed during assembly of the Mars Science Laboratory rover. Anaerobic bacteria were cultured on several media, and DNA was extracted from suitable anaerobic enrichments and examined with conventional 16S rRNA gene clone library, as well as high-density phylogenetic 16S rRNA gene microarray (PhyloChip) technologies. The culture-dependent analyses predominantly showed the presence of clostridial and propionibacterial strains. The 16S rRNA gene sequences retrieved from clone libraries revealed distinct microbial populations associated with each clean-room facility, clustered exclusively within gram-positive organisms. PhyloChip analysis detected a greater microbial diversity, spanning many phyla of bacteria, and provided a deeper insight into the microbial community structure of the clean-room facilities. This study presents an integrated approach for assessing the anaerobic microbial population within clean-room facilities, using both molecular and cultivation-based analyses. The results reveal that highly diverse anaerobic bacterial populations persist in the clean rooms even after the imposition of rigorous maintenance programs and will pose a challenge to planetary protection implementation activities.
PMCID: PMC2863428  PMID: 20228115
3.  Comprehensive Census of Bacteria in Clean Rooms by Using DNA Microarray and Cloning Methods▿ †  
Applied and Environmental Microbiology  2009;75(20):6559-6567.
A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments.
PMCID: PMC2765134  PMID: 19700540
4.  Effect of Shadowing on Survival of Bacteria under Conditions Simulating the Martian Atmosphere and UV Radiation▿ †  
Spacecraft-associated spores and four non-spore-forming bacterial isolates were prepared in Atacama Desert soil suspensions and tested both in solution and in a desiccated state to elucidate the shadowing effect of soil particulates on bacterial survival under simulated Martian atmospheric and UV irradiation conditions. All non-spore-forming cells that were prepared in nutrient-depleted, 0.2-μm-filtered desert soil (DSE) microcosms and desiccated for 75 days on aluminum died, whereas cells prepared similarly in 60-μm-filtered desert soil (DS) microcosms survived such conditions. Among the bacterial cells tested, Microbacterium schleiferi and Arthrobacter sp. exhibited elevated resistance to 254-nm UV irradiation (low-pressure Hg lamp), and their survival indices were comparable to those of DS- and DSE-associated Bacillus pumilus spores. Desiccated DSE-associated spores survived exposure to full Martian UV irradiation (200 to 400 nm) for 5 min and were only slightly affected by Martian atmospheric conditions in the absence of UV irradiation. Although prolonged UV irradiation (5 min to 12 h) killed substantial portions of the spores in DSE microcosms (∼5- to 6-log reduction with Martian UV irradiation), dramatic survival of spores was apparent in DS-spore microcosms. The survival of soil-associated wild-type spores under Martian conditions could have repercussions for forward contamination of extraterrestrial environments, especially Mars.
PMCID: PMC2258572  PMID: 18083857
5.  Isolation and Characterization of Bacteria Capable of Tolerating the Extreme Conditions of Clean Room Environments▿  
In assessing the bacterial populations present in spacecraft assembly, spacecraft test, and launch preparation facilities, extremophilic bacteria (requiring severe conditions for growth) and extremotolerant bacteria (tolerant to extreme conditions) were isolated. Several cultivation approaches were employed to select for and identify bacteria that not only survive the nutrient-limiting conditions of clean room environments but can also withstand even more inhospitable environmental stresses. Due to their proximity to spacefaring objects, these bacteria pose a considerable risk for forward contamination of extraterrestrial sites. Samples collected from four geographically distinct National Aeronautics and Space Administration clean rooms were challenged with UV-C irradiation, 5% hydrogen peroxide, heat shock, pH extremes (pH 3.0 and 11.0), temperature extremes (4°C to 65°C), and hypersalinity (25% NaCl) prior to and/or during cultivation as a means of selecting for extremotolerant bacteria. Culture-independent approaches were employed to measure viable microbial (ATP-based) and total bacterial (quantitative PCR-based) burdens. Intracellular ATP concentrations suggested a viable microbial presence ranging from below detection limits to 106 cells/m2. However, only 0.1 to 55% of these viable cells were able to grow on defined culture medium. Isolated members of the Bacillaceae family were more physiologically diverse than those reported in previous studies, including thermophiles (Geobacillus), obligate anaerobes (Paenibacillus), and halotolerant, alkalophilic species (Oceanobacillus and Exiguobacterium). Non-spore-forming microbes (α- and β-proteobacteria and actinobacteria) exhibiting tolerance to the selected stresses were also encountered. The multiassay cultivation approach employed herein enhances the current understanding of the physiological diversity of bacteria housed in these clean rooms and leads us to ponder the origin and means of translocation of thermophiles, anaerobes, and halotolerant alkalophiles into these environments.
PMCID: PMC1855582  PMID: 17308177

Results 1-5 (5)