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Archaeal Diversity Analysis of Spacecraft Assembly Clean Rooms

Christine Moissl†, James C. Bruckner, and Kasthuri Venkateswaran*

Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA91109

*Corresponding author:

Kasthuri Venkateswaran

Biotechnology and Planetary Protection Group

M/S 89, Jet Propulsion Laboratory; California Institute of Technology

4800 Oak Grove Dr., Pasadena, CA 91109

818-393-1481 Phone; 818-393-4176 Fax

Sampling locations and facilities

Samples were collected from selected surface areas within clean rooms of two distinct NASA facilities; namely, the Jet Propulsion Laboratory spacecraft assembly facility (JPL-SAF) and Johnson Space Center Genesis Curation Laboratory (JSC-GCL). Clean room certification was based on the maximum number of particles that are greater than 0.5 m per cubic foot of air (ISO 14644-1 Part 1: Classification of air cleanliness; The air within class 10 clean rooms was maintained at fewer than 10 particulates per cubic foot, class 10K clean rooms were allowed to harbor a density of 10,000 particles per cubic foot, and so on. In addition to air quality, several environmental parameters of clean rooms were also controlled, such as humidity, temperature, and air circulation. NASA quality assurance engineers performed periodic audits to ensure that certified-facility cleanliness levels conformed to the requirements delineated (La Duc et al., 2007). The air of all facilities was filtered through High Efficiency Particle Air (HEPA) filters with the exception of JSC clean rooms which utilized Ultra Low Particle Air (ULPA) filters to achieve higher cleanliness. In addition, the floors of JSC <5K certified clean rooms were raised to prevent the accumulation of organic-laden debris (La Duc et al., 2007). Details of the sampling areas and other characteristics are given in Table 1.

Sample collection

The sampling at JPL-SAF was conducted during six different times (once a month from October 2005 to April 2006 except November 2005) at three fixed locations within a class 100K clean room whereas JSC-GCL was sampled from 8 different locations of various clean rooms (four from class 10, three from class 1K, and one from uncertified room). To sample the JPL-SAF, Biological Sampling Kits (BiSKit, Quicksilver Analytics, Abingdon, MD) were used according to the manufacturer’s instructions. As the BisKiT was not certified for use in class <1K clean rooms, the JSC-GCL samples were obtained using sterile polyester wipes pre-moistened with phosphate buffered saline (PBS, pH 7.2). After sampling, wipes were placed in 50 mL tubes containing 35 mL PBS and vortexed. Samples were immediately divided into various portions and frozen (-80°C) or processed without interruption. Air sampling was done as described elsewhere (Moissl et al., 2007b).

DNA extraction, amplification of archaeal 16S rRNA genes and cloning

The JSC samples (collected using sterile wipes) were subjected to standard phenol-chloroform DNA extraction procedures (Johnson, 1981; Moissl et al., 2007). However, a modified procedure was used to extract nucleic acids from the samples collected from JPL-SAF. Briefly, sample solutions obtained from BiSKit were incubated at 65°C (30 min) after the addition of 1% sodium desoxyl sulphate (SDS). The reaction mixtures were subsequently subjected to three freeze- thaw cycles (-80°C, 65°C). After the addition of Proteinase K (Sigma-Aldrich, final concentration 500µg/ml), the samples were incubated at 37°C for 30 min. This step was followed by a phenol-chloroform extraction (Johnson, 1981) and the DNA was precipitated overnight at -20°C in 2 volumes of ice-cold ethanol. After centrifugation the ethanol was removed and the nucleic acid pellet resuspended in 50 µl sterile pure water.

In each step of the DNA extraction and PCR, suitable negative controls were used. In addition to water controls, an unopened BisKit buffer was included as a negative control in DNA extraction and PCR. All PCR reactions were performed with the following controls: a positive control (DNA of Acidianus brierleyi) and two negative controls (buffer extract and water). In order to check whether any inhibitory molecules were present in the extract all samples that exhibited negative PCR amplification for archaea were spiked with known DNA of A. brierleyi. To find the optimal amplification conditions for archaeal sequences from our samples, several preliminary tests were done with the first samples obtained (JPL October 2005). All possible primer combinations (forward: 8aF (Burggraf et al., 1992) and 345aF (Burggraf et al., 1997), reverse: 1119aR (Burggraf et al., 1997) and 1406uR (Lane, 1991)) were used for preliminary PCR reaction, as well as the nested PCR- primer combination 8aF - 1406uR, and 344aF - 1119aR. The brightest PCR signals were obtained when PCR was performed with 8aF and 1119aR, followed by a reamplification with the same primer combination. This primer combination was used for all other subsequent studies. The optimal annealing temperature for the PCR was found to be 55°C or 60°C. A 50°C annealing temperature resulted in weaker PCR signals.

The PCR was performed under the following conditions: 95°C for 4 min; 33 cycles of 95°C for 50 s, 55°C for 50 s, and 72°C for 1 min 30 s; and final incubation at 72°C for 10min. Based on the preliminary testing, reactions were performed using 2 μl of the extracted nucleic acid as a template for the amplification of archaeal 16S rDNA using the forward and reverse primers 8aF and 1119aR, respectively. The amplicons of samples and controls of the initial PCR were used as templates (2µl) for an immediate second amplification with the same primer combination. The length and quantity of the amplicons were visualized on a 1% agarose gel. Amplified PCR fragments were purified immediately (QIAquick PCR purification kit, Qiagen, Valencia, CA) and subjected to cloning using a TOPO® TA cloning kit (Invitrogen, Carlsbad, CA) according the manufacturer’s instructions. The presence of inserts of the expected size was verified by direct PCR screening of up to 120 transformants without plasmid extraction. For restriction fragment length polymorphism (RFLP) analyses, the PCR products were digested with a mixture of four different restriction endonucleases (AluI, HhaI, HinfI and RsaI; Fermentas Inc, Hanover, MD). The resulting restriction patterns were compared, and representative transformants were selected and their plasmid DNA extracted (Qiaprep kit, Qiagen, Valencia, CA) as recommended by the manufacturer. Sequencing of the 16S rRNA gene sequences was performed by Agencourt Bioscience Corporation (Beverly, MA).

Rarefaction analyses

The coverage of clone libraries was calculated according to Good (Good, 1953) using the equation: C= [1-(n1/N)]*100, where C is the homologous coverage, n1 is the number of OTU’s appearing only once in the library, and N is the total number of clones examined.

Phylogenetic analyses

All sequences obtained were submitted to the CHECK_CHIMERA program of the Ribosomal Database Project to detect possible chimeric artifacts (Cole et al., 2003). For phylogenetic analyses, an alignment of approximately 40,000 homologous full and partial sequences available in public databases was used (ARB project, (Ludwig et al., 2004)). The new 16S rRNA gene sequences were added to the 16S rRNA alignment using the corresponding automated tools of the ARB software package (Ludwig et al., 2004). The resulting alignment was checked manually and corrected if necessary. For tree reconstruction, methods were applied as implemented in the ARB software package.

Nucleotide sequence accession numbers

The 16S rRNA gene sequences of the clones were deposited in the NCBI nucleotide sequence database. The accession numbers are given in Fig. 1.

References (Supplementary Information)

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Burggraf, S., Huber, H. & Stetter, K. O. (1997). Reclassification of the crenarchael orders and families in accordance with 16S rRNA sequence data. Int J Syst Bacteriol47, 657-660.

Cole, J. R., Chai, B., Marsh, T. L. & other authors (2003). The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids Res31, 442-443.

Good, I. J. (1953). The population frequencies of species and the estimation of population parameters. Biometrika40, 237-264.

Johnson, J. L. (1981). Genetic characterization. In Manual of Methods for General Bacteriology. Edited by P. Gerhardt, R. G. E. Murray, R. N. Costilaw, E. W. Nester, W. A. Wood, N. R. Krieg & G. B. Phillips. Washington, D.C: American Society for Microbiology.

La Duc, M. T., Dekas, A. E., Osman, S., Moissl, C., Newcombe, D. & Venkateswaran, K. (2007). Isolation and characterization of bacteria capable of tolerating the extreme conditions of clean-room environments. Appl Environ Microbiol73, 2600-2611.

Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics, pp. 115-163. Edited by E. Stackebrandt & M. Goodfellow. New York, NY: John Wiley & Sons.

Ludwig, W., Strunk, O., Westram, R. & other authors (2004). ARB: a software environment for sequence data. Nucleic Acids Res32, 1363-1371.

Moissl, C., La Duc, M. T., Osman, S., Dekas, A. E. & Venkateswaran, K. (2007). Molecular bacterial community analysis of clean rooms where spacecraft are assembled. FEMS Microbiol Ecol (in press)61, 509-521.

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