New perspectives on viable microbial communities in low-biomass cleanroom environments.

The advent of phylogenetic DNA microarrays and high-throughput pyrosequencing technologies has dramatically increased the resolution and accuracy of detection of distinct microbial lineages in mixed microbial assemblages. Despite an expanding array of approaches for detecting microbes in a given sample, rapid and robust means of assessing the differential viability of these cells, as a function of phylogenetic lineage, remain elusive. In this study, pre-PCR propidium monoazide (PMA) treatment was coupled with downstream pyrosequencing and PhyloChip DNA microarray analyses to better understand the frequency, diversity and distribution of viable bacteria in spacecraft assembly cleanrooms. Sample fractions not treated with PMA, which were indicative of the presence of both live and dead cells, yielded a great abundance of highly diverse bacterial pyrosequences. In contrast, only 1% to 10% of all of the pyrosequencing reads, arising from a few robust bacterial lineages, originated from sample fractions that had been pre-treated with PMA. The results of PhyloChip analyses of PMA-treated and -untreated sample fractions were in agreement with those of pyrosequencing. The viable bacterial population detected in cleanrooms devoid of spacecraft hardware was far more diverse than that observed in cleanrooms that housed mission-critical spacecraft hardware. The latter was dominated by hardy, robust organisms previously reported to survive in oligotrophic cleanroom environments. Presented here are the findings of the first ever comprehensive effort to assess the viability of cells in low-biomass environmental samples, and correlate differential viability with phylogenetic affiliation.

The C. elegans rab family: identification, classification and toolkit construction.

Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).