A microarray for assessing transcription from pelagic marine microbial taxa

The ISME Journal (2014) 8, 1476–1491 & 2014 International Society for Microbial Ecology All rights reserved 1751-7362/14 www.nature.com/ismej ORIGINAL ARTICLE A microarray for assessing transcription from pelagic marine microbial taxa Irina N Shilova1, Julie C Robidart1, H James Tripp2, Kendra Turk-Kubo1, Boris Wawrik3, Anton F Post4, Anne W Thompson5, Bess Ward6, James T Hollibaugh7, Andy Millard8, Martin Ostrowski8, David J Scanlan8, Ryan W Paerl9, Rhona Stuart10 and Jonathan P Zehr1 1 Department of Ocean Sciences, University of California Santa Cruz, Santa Cruz, CA, USA; 2DOE Joint Genome Institute, Walnut Creek, CA, USA; 3Department of Microbiology and Plant Biology, University of Oklahoma, Oklahoma, USA; 4Marine Biological Laboratory, Woods Hole, MA, USA; 5Advanced Cytometry Group, BD Biosciences, Seattle, WA, USA; 6Department of Geosciences, Princeton University, Princeton, NJ, USA; 7Department of Marine Sciences, University of Georgia, Athens, GA, USA; 8Department of Marine Microbiology, University of Warwick, Coventry, UK; 9Marine Biology Research Division, University of California San Diego, San Diego, CA, USA and 10Physical and Life Sciences, Lawrence Livermore National Laboratory, Livermore, CA, USA 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. The ISME Journal (2014) 8, 1476–1491; doi:10.1038/ismej.2014.1; published online 30 January 2014 Subject Category: Integrated genomics and post-genomics approaches in microbial ecology Keywords: marine; microbial; microarray; transcription; molecular Introduction Marine microbial communities are complex, composed of diverse groups of Bacteria, Archaea, Eukaryotes and viruses. Molecular techniques frequently used in marine microbial ecology have shown strainspecific differences in genetic capabilities and transcriptional responses among the most abundant representatives of microbial communities (Fuhrman et al., 2006; Giovannoni and Vergin, 2012). Clades of Correspondence: IN Shilova, Department of Ocean Sciences, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA 95064, USA. E-mail: Received 29 May 2013; revised 16 December 2013; accepted 31 December 2013; published online 30 January 2014 Prochlorococcus in the North Atlantic have more phosphorus-acquisition strategies than clades in the North Pacific, as an adaptation to chronic phosphate limitation (Coleman and Chisholm, 2010). Coastal clades of Synechococcus have higher number of regulatory systems and the use for metals than open ocean clades, the latter being adapted to relatively constant oligotrophic conditions (Palenik et al., 2006). To link ocean processes to microbial metabolism and to build better models for predicting responses to future ocean states (Azam and Malfatti, 2007), in light of this strain-level heterogeneity, new research tools are needed that assess individual and microbial community responses. Microarray technology can complement more commonly used molecular techniques, such as PCR and MicroTOOLs microarray for marine microbial taxa IN Shilova et al 1477 next-generation sequencing, to provide cost-effective high-throughput gene and transcript detection from several organisms in a single sample. Microarrays have the advantages of sample replication, standardization and robust interpretations of strain-level variation in functional gene transcriptional patterns and lend themselves to better comparative quantification of specific genes and transcripts, especially in rare organisms. Phylogenetic and functional microarrays have been developed and used for identification of microorganisms and their activity in diverse environments. The most comprehensive microbial functional microarray to date, the GeoChip 4.0, targets 410 000 sequences represented by 150 genes mainly from soil microbial communities and reduction– oxidation processes (He et al., 2007, 2010; Bai et al., 2013; Zhou et al., 2013). In addition to contaminated soils, acid mine drainage sites and Antarctic soils (Yergeau et al., 2007; Mason et al., 2010; Xie et al., 2011), the GeoChip has been applied to detect microbial DNA and RNA in the marine environment (Lu et al., 2012; Wawrik et al., 2012). Aside from the PhyloChip, which targets 16S rRNA genes (Brodie et al., 2006), existing microarrays target a specific genus (Rinta-Kanto et al., 2011), a particular process (Tiquia et al., 2004; Moisander et al., 2006, 2007; Ward et al., 2007; Bulow et al., 2008; Wu et al., 2008; Bouskill et al., 2011; Abell et al., 2012) or ecosystem (Rich et al., 2008; Smith et al., 2010; Rich et al., 2011). A comprehensive microarray that targets functional genes across diverse pelagic marine microbial communities has not yet been reported. Various strategies have been used to overcome the lack of a priori knowledge of genomic sequences in target communities, which is a major limitation in the design of an environmental microarray. The most common strategy is to search public nucleotide (nt) sequence databases (such as National Center for Biotechnology Information (NCBI)) using key words (Rhee et al., 2004; He et al., 2007; Wu et al., 2008; He et al., 2010; Smith et al., 2010). The resulting data sets, however, typically do not resemble the natural diversity of target genes, a problem that is exacerbated in undersampled environments. A second common strate (...truncated)