Project description:Microarrays are useful tools for detecting and quantifying specific functional and phylogenetic genes in natural microbial communities. In order to track uncultivated microbial genotypes and their close relatives in an environmental context, we designed and implemented a “genome proxy” microarray that targets microbial genome fragments recovered directly from the environment. Fragments consisted of sequenced clones from large-insert genomic libraries from microbial communities in Monterey Bay, the Hawaii Ocean Time-series station ALOHA, and Antarctic coastal waters. In a prototype array, we designed probe sets to thirteen of the sequenced genome fragments and to genomic regions of the cultivated cyanobacterium Prochlorococcus MED4. Each probe set consisted of multiple 70-mers, each targeting an individual ORF, and distributed along each ~40-160kbp contiguous genomic region. The targeted organisms or clones, and close relatives, were hybridized to the array both as pure DNA mixtures and as additions of cells to a background of coastal seawater. This prototype array correctly identified the presence or absence of the target organisms and their relatives in laboratory mixes, with negligible cross-hybridization to organisms having ≤~75% genomic identity. In addition, the array correctly identified target cells added to a background of environmental DNA, with a limit of detection of ~0.1% of the community, corresponding to ~10^3 cells/ml in these samples. Signal correlated to cell concentration with an R2 of 1.0 across six orders of magnitude. In addition the array could track a related strain (at 86% genomic identity to that targeted) with a linearity of R2=0.9999 and a limit of detection of ~1% of the community. Closely related genotypes were distinguishable by differing hybridization patterns across each probe set. This array’s multiple-probe, “genome-proxy” approach and consequent ability to track both target genotypes and their close relatives is important for the array’s environmental application given the recent discoveries of considerable intra-population diversity within marine microbial communities. Keywords: target addition experiment, proof-of-concept for GPL6012

Project description:Marine snow plays a central role in carbon cycling. It consists of organic particles and particle-associated (PA) microbMarine snow plays a central role in carbon cycling. It consists of organic particles and particle-associated (PA) microbial communities that are embedded in a sugary matrix. Metaproteomic analysis offers the unique opportunity to gain unprecedented insight into the microbial community composition and biomolecular activity of environmental samples. In order to realize this potential for marine PA microbial communities, new methods of protein extraction must be developed. In this study, we used 1D-SDS-PAGEs and LC-MS/MS to compare the efficiency of six established protein extraction protocols for their applicability of metaproteomic analyses of the PA microbial community in the North Sea. A combination of SDS-buffer extraction and bead beating resulted in the greatest number of identified protein groups. As expected, a metagenomic database of the same environmental sample increased the number of protein identification by approximately 50%. To demonstrate the application of our established protocol, particulate bacterioplankton samples collected during spring phytoplankton bloom in 2009 near the island Helgoland, were analysed by a GeLC-MS/MS-based metaproteomic approach. Our results indicated that there are only slight differences in the taxonomical distribution between free-living (FL) and PA bacteria but that the abundance of protein groups involved in polysaccharide degradation, motility and particle specific stress (oxygen limitation, nutrient limitation, heavy metal stress) is higher in the PA fractions. ial communities that are embedded in a sugary matrix. Metaproteomic analysis offers the unique opportunity to gain unprecedented insight into the microbial community composition and biomolecular activity of environmental samples. In order to realize this potential for marine PA microbial communities, new methods of protein extraction must be developed. In this study, we used 1D-SDS-PAGEs and LC-MS/MS to compare the efficiency of six established protein extraction protocols for the their applicability of metaproteomic analyses of the PA microbial community in the North Sea. A combination of SDS-buffer extraction and bead beating resulted in the greatest number of identified protein groups. As expected, a metagenomic database of the same environmental sample increased the number of protein identification by approximately 50%. To demonstrate the application of our established protocol, particulate bacterioplankton samples collected during spring phytoplankton bloom in 2009 near the island Helgoland, were analysed by a GeLC-MS/MS-based metaproteomic approach. Our results indicated that there are only slight differences in the taxonomical distribution between free-living (FL) and PA bacteria but that the abundance of protein groups involved in polysaccharide degradation, motility and particle specific stress (oxygen limitation, nutrient limitation, heavy metal stress) is higher in the PA fractions.

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Microbial communities marine hydrothermal vents

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