TOTAL MICROBIAL COMMUNITY ANALYSES
Until recently, the ability to monitor microbial community structure was limited by the lack of suitable means to define species composition and the relative abundance of specific populations in microbial communities. Efforts to do these things have historically relied on culture-dependent methods (11, 24, 32, 44, 72) that are intrinsically limited because the vast majority of bacterial populations in natural communities are refractory to cultivation (33). The accepted protocols for determining microbial burdens on spacecraft surfaces have not been changed in 25 years. The NASA standard assay (31) is used for enumeration of spores and heterotrophic microbial populations. This assay is based on viable counting techniques, such as washing of surfaces with a sterile phosphate-buffered rinse solution with mild sonication, after which the rinse solution is aseptically analyzed for numbers of microbes by standard pour plate techniques using media such as tryptic soy agar. These protocols clearly cannot access the large number of microorganisms in most environments, including spacecraft surfaces, that are presently uncultivable using standard media. For this reason attempts to characterize the structure of even a single community can at best provide only a biased and incomplete view. Moreover, these methods are extremely laborious, and so for practical reasons it has been virtually impossible to do extensive studies to assess changes in communities over time, between locations, or in response to changes in environmental conditions.
There has been some progress in devising means to isolate some previously uncultivated bacteria. For example, Kaeberlein et al. (33) designed diffusion chambers that allowed the growth of previously uncultivated microorganisms in a simulated natural environment. These isolates did not grow on artificial media alone but formed colonies in the presence of other microorganisms. Although this method is less likely to produce results for dry and sparse spacecraft assembly facility communities, as opposed to the marine communities used by Kaeberlein et al. (33), it is a straightforward approach that could be employed in other systems. Stevenson et al. (63) used what they termed an integrative approach to obtain pure cultures of previously uncultivated Acidobacteria and Verrucomicrobia from agricultural soil and from the guts of wood-feeding termites. The techniques used included the use of agar media with little or no added nutrients, long periods of incubation (>30 days), protection of cells from peroxides, and inclusion of humic acids or a humic acid analogue and quorum-signaling compounds in growth media. However, even the approaches of Kaeberlein et al. (33) and Stevenson et al. (63) ultimately can allow observation of only a very small fraction of the types of bacterial species that are actually present in nature, which probably number in the millions (20).
Fortunately, these limitations on characterization of microbial communities have been overcome to a significant degree through the development of methods that do not rely on cultivation of microbial populations but instead are based on analysis of 16S and 18S rRNA gene sequences that are found in all living organisms. In recent years these methods have been widely employed to explore microbial diversity in diverse microbial habitats and to characterize organisms that have not been cultured yet. The basic strategy has been to use total DNA isolated from a microbial community (metagenomic DNA) as a template for PCR amplification of 16S and 18S rRNA genes using universal primers or primers that are specific for various phylogenetic domains. This is typically followed by construction of an rRNA gene clone library and analysis of individual rRNA gene clones by sequencing or by assessment of restriction fragment length polymorphisms (14) and subsequently determining the phylogeny of the constituent populations. Liu et al. (38) developed a technique called terminal restriction fragment length polymorphism analysis that extends and simplifies this approach by obviating the need to construct a clone library. Briefly, rRNA genes are obtained from total community DNA as described above, except that one of the primers used is labeled with a fluorescent dye. The mixture of rRNA genes is then digested with restriction enzymes that have 4-bp recognition sites, and the size and relative abundance of each fluorescently labeled terminal restriction fragment are determined using an automated DNA sequencer. Since a single fragment represents each numerically dominant member of the community, nominal estimates of diversity within communities can readily be obtained. The pattern of terminal restriction fragments observed (referred to as the "community fingerprint") is a composite of the number of fragments with unique lengths, and the relative abundance of each fragment is roughly reflected in the size of each peak in the electropherogram. Other techniques for microbial community profiling include denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, single-strand conformation polymorphism analysis, amplified rRNA gene restriction analysis, and amplified intergenic spacer analysis (2).
An assessment of microbial diversity within spacecraft assembly facilities using these modern tools is critically needed to provide basic knowledge concerning the diversity of microorganisms that might contaminate spacecraft that visit non-Earth planetary systems. Less than 1% of the microorganisms present in most natural environments have been grown in pure culture (33). This is almost certainly also true of spacecraft assembly facilities, as the preliminary data of Venkateswaran et al. (69, 70) indicate. Current international treaties and United States regulations require enumeration of cultivable microorganisms but not identification. Previous NASA studies of cultivable strains have provided only a partial and inadequate picture of the microbial community structure within spacecraft assembly facilities. As has been the case in Antarctica (40), there are concerns of possible anthropogenic contamination of Mars, Titan, Europa, and other locations with Earth-derived organic contaminants or life forms that might (i) colonize or survive in their new location and/or (ii) complicate later searches for extraterrestrial life forms. The former is possible since the microbes of spacecraft assembly facilities are known to be highly adapted to extreme environmental conditions, even those that might be encountered on other planets or their moons (69, 70). The latter is possible since the most environmentally robust life forms that may have evolved in extraterrestrial locations may be, as on Earth, microbial. It will be difficult to exclude Earth-derived contamination as a source of observed microbes if we do not thoroughly understand the complete composition of communities that spacecraft might inadvertently deliver.
Colleagues and I examined the potential for survival of B. subtilis endospores on Mars that could some day be inadvertently delivered to that planet as contaminants aboard a spacecraft (15). Mars, which is thought to have highly oxidizing soil, is considered by many researchers to be completely inhospitable to life as we know it on Earth. If so, this would make delivery of Earth microbes and even spores to Mars of little concern. Thus, we examined the hypothesis that if the soil of Mars contains iron as ferrate VI (26), it is self-sterilizing. Ferrate is one of the strongest oxidants known and exists on Earth only in the laboratory. We incubated dried endospores of B. subtilis in a Mars surrogate soil comprised of dry silica sand containing 20% (by weight) synthetic ferrate dianion (FeO42–), but we used incubation conditions similar to those present on Mars. These conditions included extreme desiccation, high levels of surface UV radiation, cold, and a CO2-dominated atmosphere. The endospores were not killed and were very resistant to inactivation by the oxidant-enriched sand, even in the presence of high fluxes of sterilizing UV radiation, as long as they were protected by a shallow layer of sand (15). Similar results were observed with permanganate, another strong oxidant. Ronto et al. (55) pointed out that the current Martian UV environment is still quite severe from a biological viewpoint but also showed that substantial protection can be afforded to microbial spores under dust and ice. Based on these data and previously published (controversial) descriptions of ancient but dormant life forms on Earth (13, 22, 27, 28, 43, 71), we concluded that if highly resistant endospores such as those studied at JPL (36, 68, 69) were delivered to Mars, they may remain viable for many years or even indefinitely. Considering the spores within the JPL spacecraft assembly facility that we know about, which are far more robust than B. subtilis spores, we clearly need to understand the true diversity of all microbial forms present, even the forms we cannot grow.
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