While molecular biology has seen monumental advances in the specificity and sensitivity of modern techniques, the efficient collection and accurate phylogenetic analysis of microorganisms from low-biomass samples remain extremely challenging (19
). Since the effective sampling area of a spacecraft is fixed, it is not possible simply to increase the sample size to improve yield (4
). It is of utmost importance to ensure that current methods of assessing phylogenetic breadth and overall microbial burden from these precious allotments are optimal for conserving the true microbial community structure of the sampled environment. Therefore, as reported previously (19
), considerable measures were taken to ensure that optimized sample collection and automated sample processing procedures were integrated so as to elucidate the fullest possible spectrum of microbial life associated with spacecraft surfaces. Given the constraints inherent to working with such low-biomass samples, technologies capable of accurately registering low-abundance organisms are vital.
The rapidity, repeatability, comprehensiveness, and sensitivity of the PhyloChip for surveying entire bacterial communities in environmental samples suggest that the approach could significantly advance microbial detection and environmental monitoring. Key features that set the PhyloChip apart from similar technologies are the use of multiple oligonucleotide probes for all known prokaryotic taxa for high-confidence detection and the pairing of a mismatch probe for every perfectly matched probe to minimize the effect of nonspecific hybridization (35
). A strong linear correlation has been confirmed between microarray probe set intensity and concentration of OTU-specific 16S rRNA gene copies, allowing quantification in a broad dynamic range. Validation experiments have demonstrated high reproducibility as intensity responses among replicate chips show less than 10% variation (3
). PhyloChip results from complex environmental samples have been confirmed by additional methods, including quantitative PCR and 16S rRNA gene clone libraries (7
), and analyses of split samples have confirmed that >90% of all 16S rRNA sequence types identified by the more expensive clone library method are also identified by the PhyloChip. When the high-density PhyloChip microarray, with all known DNA sequences encoding bacterial and archaeal 16S rRNA (9
), was applied to urban aerosols, the spatiotemporal distributions of known bacterial groups, including specific pathogens, were related to meteorologically driven transport processes as well as sources (12
Previous analyses of surface samples collected at three different time periods (before, during, and after Phoenix Lander assembly/occupancy) from the same locations within the KSC-PHSF clean room led to the conclusion that cleaning protocols in use were indeed effective in significantly reducing both microbial burden (13
) and diversity (P. Vaishampayan et al., submitted). As might be expected, the clone libraries representing the pre-Phoenix sampling (PHSF-PHX-B, where PHX-B indicates before Phoenix Lander assembly/occupancy) exhibited a great many OTUs (166 OTUs by cloning), and the corresponding coverage value was low (86.5%). However, with increased cleaning efforts during (Table , PHX-D1) and after (PHX-A) Phoenix, detectable OTUs were significantly reduced (~20), and coverage values escalated to ~92%. Such a trend was also observed with samples collected before (MSL-B; 76% coverage), and during (MSL-D1; 93% coverage) MSL occupancy of the JPL-SAF. These observed reductions in bacterial numbers while facilities were housing spacecraft (Table , coverage values) can likely be attributed to more diligent cleaning efforts as the frequency of cleaning increased (two- to threefold increase in schedule) when spacecraft were present, as opposed to standard facility maintenance during nonoperational periods (twice per week). Immediately following the departure of the Phoenix spacecraft from the KSC-PHSF, the facility was maintained at utmost stringency, and no changes were made in cleaning practices so that the facility would be ready to accommodate any unforeseen needs associated with a launch delay. Samples collected at this time (post-Phoenix with bolstered cleaning and maintenance) continued to exhibit appreciable coverage values (98.5%) even though the spacecraft was not present.
Perhaps the greatest advantage of cloning-based biodiversity analysis was the ability to generate rarefaction curves and corresponding coverage values, which provided an invaluable approximation of just how representative each sample was of its true environment (31
). Due in large part to biases in the generation and picking of transformant colonies, PhyloChip DNA microarrays detected a much broader biodiversity than clone libraries, even at very high taxonomic levels (7
). There was an appreciable difference in the level at which the PhyloChip “out-detected” cloning approaches, based on the presence or absence of spacecraft hardware at the time of sample collection. The superior detection capabilities of the PhyloChip were far more pronounced when the facility was sampled while housing spacecraft hardware (32- to 70-fold) than when sampled facilities sat vacant (9- to 16-fold). This was a reasonable correlation since the bacterial diversity associated with any given SAC should be a combination of the bacterial diversities associated with that facility plus that associated with foreign spacecraft components that have been fabricated from countless geographic locations.
Compared directly, MSL-supporting SAC samples did not house as rich a diversity of bacteria as samples collected from facilities housing Phoenix hardware. This is not to say that MSL-associated SAC were not diverse. DNA microarray analyses detected roughly 4,000 OTUs in the five MSL-associated SAC samples; however, only ca. 150 OTUs were detected in all five of these samples. This is of immense consequence for planetary protection and/or the validation of clean room maintenance as it suggests that frequent monitoring is required over the course of a project or process to confidently assess the majority of contaminant microbes associated with production/assembly facility surfaces (and therefore at risk of being sent into space on spacecraft).
The systematic approach taken during this study revealed that the PhyloChip microarray analyses were superior to conventional 16S rRNA gene cloning and sequencing strategies in all aspects of microbial diversity analysis save one: the detection of novel microbial taxa. Since DNA microarrays are dependent on the hybridization of environmental oligonucleotides to known probes of specific sequence, an enormous amount of a priori sequence information is required. This need for previously inferred probe sequence data precludes the ability of this technique to detect the presence of DNA arising from novel microorganisms. As shown in Fig. , there were a few novel taxa whose presence completely eluded the PhyloChip but was inferred from clone library analysis alone. As for limitations, with the cloning and mass sequencing approach there was likely a molecular bias that favored the PCR amplification and/or amplicon ligation of certain bacterial lineages and hence masked the detection of taxa that were present in much lower abundance. High-throughput approaches possess a significant advantage to cloning in that they are much more capable of yielding valuable phylogenetic information from samples (7
). Ultimately, PhyloChip DNA microarray analyses supported, and accentuated, the general trends observed by clone libraries with regard to geographic clustering (data not shown). The results of this comparative study underscore a central theme in current molecular biology: a shift toward high-throughput, data-rich molecular assays requiring significant bioinformatics analysis.
There are numerous factors to consider in choosing an appropriate methodology for elucidating microbial diversity in environmental samples. While factors of cost, time, labor intensity, and reproducibility weigh quite heavily individually, the bias and accuracy of a given approach are perhaps the most important aspects in considering the goal of planetary protection endeavors. In an effort to significantly strengthen the inferences drawn from extraterrestrial life detection experiments, NASA has stressed the importance of taking necessary precautions to ensure that spacecraft outbound from Earth are as devoid of microbial contaminants as reasonably possible. One approach to achieving this objective is to routinely survey and catalog the genetic microbial inventory present on SAC and colocated spacecraft surfaces. These efforts will prove invaluable in interpreting the findings of numerous robotic extraterrestrial life detection missions. By working to minimize the microbial burden associated with robotic spacecraft to levels approaching near sterility and routinely sampling from and maintaining a genetic inventory of the microbes associated with spacecraft and SAC, planetary protection efforts are (i) minimizing the likelihood that life detection experiments will be compromised by contaminant terrestrial biomatter, (ii) increasing the ability to discriminate authigenic from contaminant biomaterial should any be detected, and (ii) benefitting a wide range of scientific, electronic, homeland security, medical, and pharmaceutical ventures by developing superior means of detecting and mitigating microbial contaminants from low-biomass environments.