Project description:Metagenomic approaches have revealed unprecedented genetic diversity within microbial communities across vast expanses of the world’s oceans. Linking this genetic diversity with key metabolic and cellular activities of microbial assemblages is a fundamental challenge. Here we report on a collaborative effort to design MicroTOOLs (Microbiological Targets for Ocean Observing Laboratories), a high-density oligonucleotide microarray that targets functional genes of diverse taxa in pelagic and coastal marine microbial communities. MicroTOOLs integrates nucleotide sequence information from disparate data types: genomes, PCR-amplicons, metagenomes, and metatranscriptomes. It targets 19 400 unique sequences over 145 different genes that are relevant to stress responses and microbial metabolism across the three domains of life and viruses. MicroTOOLs was used in a proof-of-concept experiment that compared the functional responses of microbial communities following Fe and P enrichments of surface water samples from the North Pacific Subtropical Gyre. We detected transcription of 68% of the gene targets across major taxonomic groups, and the pattern of transcription indicated relief from Fe limitation and transition to N limitation in some taxa. Prochlorococcus (eHLI), Synechococcus (sub-cluster 5.3) and Alphaproteobacteria SAR11 clade (HIMB59) showed the strongest responses to the Fe enrichment. In addition, members of uncharacterized lineages also responded. The MicroTOOLs microarray provides a robust tool for comprehensive characterization of major functional groups of microbes in the open ocean, and the design can be easily amended for specific environments and research questions.
Project description:Increasing atmospheric CO2 concentrations are causing decreased pH over vast expanses of the ocean. This decreasing pH may alter biogeochemical cycling of carbon and nitrogen via the microbial process of nitrification, a key process that couples these cycles in the ocean, but which is often sensitive to acidic conditions. Recent reports indicate a decrease in oceanic nitrification rates under experimentally lowered pH. How composition and abundance of ammonia oxidizing bacteria (AOB) and archaea (AOA) assemblages respond to decreasing oceanic pH, however, is unknown. We sampled microbes from two different acidification experiments and used a combination of qPCR and functional gene microarrays for the ammonia monooxygenase gene (amoA) to assess how acidification alters the structure of ammonia oxidizer assemblages. We show that despite widely different experimental conditions, acidification consistently altered the community composition of AOB by increasing the relative abundance of taxa related to the Nitrosomonas ureae clade. In one experiment this increase was sufficient to cause an increase in the overall abundance of AOB. There were no systematic shifts in the community structure or abundance of AOA in either experiment. These different responses to acidification underscore the important role of microbial community structure in the resiliency of marine ecosystems. amoA gene diversity from two ocean acidification experiments, Monterey Bay experiment (two time points, ambient and acidified) and Vineyard Sound experiment (ambient and acifidied, with and without nutrients) examined with 2 two-color arrays (Cy3 and Cy5): the universal standard 20-mer oligo is printed to the slide with a 70-mer oligo (an archetype). Environmental DNA sequences (fluoresced with Cy3) within 15% of the 70-mer conjugated to a 20-mer oligo (fluoresced with Cy5) complementary to the universal standard will bind to the oligo probes on the array. Signal is the ratio of Cy3 to Cy5.
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