Analysis of sulfur-related transcription by Roseobacter communities using a taxon-specific functional gene microarray

The fraction of dissolved dimethylsulfoniopropionate (DMSPd) converted by marine bacterioplankton into the climate-active gas dimethylsulfide (DMS) varies widely in the ocean, with the factors that determine this value still largely unknown. One current hypothesis is that the ratio of DMS formation:DMSP demethylation is determined by DMSP availability, with 'availability' in both an absolute sense (i.e., concentration in seawater) and in a relative sense (i.e., proportionally to other labile organic S compounds) being proposed as the critical factor. We investigated these models during an experimentally-induced phytoplankton bloom using an environmental microarray targeting DMSP-related gene expression in the Roseobacter group, a taxon of marine bacteria known to play an important role in the surface ocean sulfur cycle. The array consisted of 1,578 probes to 431 genes, including those previously linked to DMSP degradation as well as core genes common in sequenced Roseobacter genomes. The prevailing pattern of Roseobacter gene expression showed depletion of DMSP-related transcripts during the peak of the bloom, despite the fact that absolute concentrations and flux of DMSP-related compounds were increasing. A likely interpretation is that DMSPd was assimilated by Roseobacter populations in proportion to its relative abundance in the organic matter pool (the “relative sense” hypothesis), and that it is not taken up in preference to other sources of labile organic sulfur or carbon produced during the bloom. The relative investment of the Roseobacter community in DMSP demethylation did not predict the fractional conversion of DMSP to DMS, however, suggesting a complex regulatory process that may involve multiple fates of DMSPd. DMSP-related gene expression in the Roseobacter group was investigated using an environmental microarray. Coastal seawater from the Gulf of Mexico was collected and dispensed into 20-L microcosms. Two replicate cubitainers were amended with nutrients (N and P) to stimulate phytoplankton bloom, while two untreated cubitainers served as controls. The microcosms were incubated at 27ºC in a temperature-controlled incubator on a 12 h light/dark cycle for total of 7 days. Ten RNA samples (Day 0: 2 conditions with 1 biological replicate each; Days 2 and 4: 2 conditions with 2 biological replicates each) were prepared for microarray hybridization. After total RNA extraction, rRNAs were removed and mRNA transcripts were amplified and labeled with Alexa Fluor 647. Two technical replicates were hybridized from each RNA sample. The microarray was designed based on selected Ruegeria pomeroyi DSS-3 genes and their orthologs in 12 other sequenced Roseobacter genomes. Probes were designed from the orthologs using the Hierarchical Probe Design (HPD) algorithm.