Comprehensive census of bacteria in clean rooms by using DNA microarray and cloning methods

Abstract

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.

FIG. 1.

Family level phylogenetic tree of bacterial taxa detected across all sampling events using PhyloChip technologies, clone library sequencing, or both. The unclassified bacterial groups (∼30) were not included in the construction of this phylogenetic tree. Bacterial groups in which the PhyloChip detected significant differences in biodiversity between the JPL-SAF and the KSC-PHSF are noted (†) and described further in Table 2.

FIG. 2.

Bacterial families detected across various SAC categories, as defined in the text.

FIG. 3.

PhyloChip analysis of complete bacterial communities as a function of SAC categorization. Bacteria are ordered alphabetically from left to right according to taxonomic affiliation. Bars above the zero line represent bacteria that increased in abundance relative to the JPL-SAF during the MSL mission; bars below represent those bacteria that declined in abundance. Venn diagrams demonstrate the number of bacterial subfamilies detected in each SAC category. proteo, proteobacteria.