The ISME Journal (2013) 7, 1775–1789 & 2013 International Society for Microbial Ecology All rights reserved 1751-7362/13 www.nature.com/ismej ORIGINAL ARTICLE Temporal metatranscriptomic patterning in phototrophic Chloroflexi inhabiting a microbial mat in a geothermal spring Christian G Klatt1,6, Zhenfeng Liu2, Marcus Ludwig2, Michael Ku¨ hl3,4, Sheila I Jensen3, Donald A Bryant2,5 and David M Ward1 1Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, MT, USA; 2Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA; 3Marine Biology Section, Department of Biology, University of Copenhagen, Helsingør, Denmark; 4Plant Functional Biology and Climate Change Cluster, Department of Environmental Science, University of Technology, Sydney, New South Wales, Australia and 5Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, USA Filamentous anoxygenic phototrophs (FAPs) are abundant members of microbial mat communities inhabiting neutral and alkaline geothermal springs. Natural populations of FAPs related to Chloroflexus spp. and Roseiflexus spp. have been well characterized in Mushroom Spring, where they occur with unicellular cyanobacteria related to Synechococcus spp. strains A and B0. Metatranscriptomic sequencing was applied to the microbial community to determine how FAPs regulate their gene expression in response to fluctuating environmental conditions and resource availability over a diel period. Transcripts for genes involved in the biosynthesis of bacteriochlor- ophylls (BChls) and photosynthetic reaction centers were much more abundant at night. Both Roseiflexus spp. and Chloroflexus spp. expressed key genes involved in the 3-hydroxypropionate (3-OHP) carbon dioxide fixation bi-cycle during the day, when these FAPs have been thought to perform primarily photoheterotrophic and/or aerobic chemoorganotrophic metabolism. The expression of genes for the synthesis and degradation of storage polymers, including glycogen, polyhydroxyalkanoates and wax esters, suggests that FAPs produce and utilize these compounds at different times during the diel cycle. We summarize these results in a proposed conceptual model for temporal changes in central carbon metabolism and energy production of FAPs living in a natural environment. The model proposes that, at night, Chloroflexus spp. and Roseiflexus spp. synthesize BChl, components of the photosynthetic apparatus, polyhydroxyalkanoates and wax esters in concert with fermentation of glycogen. It further proposes that, in daytime, polyhydrox- yalkanoates and wax esters are degraded and used as carbon and electron reserves to support photomixotrophy via the 3-OHP bi-cycle. The ISME Journal (2013) 7, 1775–1789; doi:10.1038/ismej.2013.52; published online 11 April 2013 Subject Category: Integrated genomics and post-genomics approaches in microbial ecology Keywords: filamentous anoxygenic phototrophs; metagenomics; metatranscriptomics; mixotrophy; phototrophy Introduction systems for exploring general ecological principles in microbial ecology (Ward et al., 2008). Molecular Microbial mats in the effluent channels of the characterization of thermophilic microbial mat alkaline siliceous Octopus Spring and Mushroom communities thriving in these springs at 50–74 1C Spring (Yellowstone National Park, USA) are among revealed that chlorophototrophs are the dominant the most intensively studied natural microbial community members. Specifically, the most abun- communities (Ward et al., 2006) and serve as model dant organisms consist of cyanobacteria related to cultivated Synechococcus spp. strains A and B0 (Ward et al., 1990; Ferris et al., 1996; Allewalt et al., Correspondence: CG Klatt, Department of Forest Ecology and Management, Swedish University of Agricultural Sciences, 2006; Bhaya et al., 2007) and filamentous anoxy- Skogsmarksgra¨nd, Umea˚,Va¨sterbotten SE-90183, Sweden. genic phototrophs (FAPs) related to Roseiflexus spp. E-mail: [email protected] and Chloroflexus spp. (Nu¨ bel et al., 2002; van der 6Current address: Department of Forest Ecology and Management, Meer et al., 2010). Past work has suggested that Swedish University of Agricultural Sciences, SE-90183 Umea˚, Sweden. Synechococcus spp. are the primary producers Received 26 September 2012; revised 10 February 2013; accepted responsible for most inorganic carbon fixation. 13 February 2013; published online 11 April 2013 They also produce low-molecular weight organic Metatranscriptomics of Chloroflexi CG Klatt et al 1776 compounds as byproducts of their metabolism, and community members might escape competition for it has been shown that FAPs assimilate these resources and enable coexistence in these mats by compounds photoheterotrophically (Sandbeck and temporally partitioning nutrient uptake. Ward 1981; Anderson et al. 1987; Bateson and Ward In this study, metatranscriptomic sequencing of 1988; Nu¨ bel et al., 2002). Metabolites excreted by RNA extracted from mat samples taken hourly over cyanobacteria in these mats fluctuate between day- the course of a diel period was performed. The data time production of glycolate (a byproduct of photo- provided a more complete view of how chloropho- respiration under conditions of O2 supersaturation totrophic members of the Chloroflexi temporally during the day; Bateson and Ward 1988) and transcribe their genes in relation to changing nighttime production of acetate and propionate in situ environmental conditions and the metabolic (both produced, in part, by cyanobacterial or other activities of other community members. This bacterial fermentation under anoxic conditions; experiment provided high-resolution, temporal Anderson et al., 1987, Nold and Ward 1996, van transcription profiles of genes involved in photo- der Meer et al., 2005, 2007). FAPs are thought to synthesis, central carbon metabolism and energy perform photoheterotrophic metabolism for the production of uncultivated FAPs in their natural uptake of low-molecular weight carbon sources both habitat. From these transcriptional analyses, a in culture and in situ (Madigan et al., 1974; Pierson model is proposed to describe how members of and Castenholz, 1974; Sandbeck and Ward, 1981; the Chloroflexi regulate their metabolism and con- Anderson et al., 1987; Hanada et al., 2002; van der tribute to the metabolic activities of the microbial Meer et al., 2003; van der Meer et al., 2005, 2010). mat community. Given that Chloroflexus aurantiacus strain OK-70-fl can be grown photoautotrophically on a minimal medium sparged with H2 and with CO2 as the sole Materials and methods source of carbon (Holo and Sireva˚g 1986; Strauss et al., 1992), it is of interest whether, and when, Sampling and sequence construction uncultivated phototrophic Chloroflexi can perform RNA was extracted and cDNA sequences were inorganic carbon fixation. There is evidence that produced from microbial mat samples taken at hourly Roseiflexus spp. and Chloroflexus spp. in these mats intervals from the 60 1C region of the effluent channel might fix inorganic carbon in situ when light and of Mushroom Spring over a 24-h period beginning at electron donors, such as H2 and H2S, are available at 1700 hours on 11 September 2009 and ending at 1600 dawn and possibly at dusk (van der Meer et al., hours the following day (Liu et al., 2012). A processing 2005; Klatt et al., 2007). Furthermore, the 3-hydro- error caused the sample for 1100 hours to be lost. The xypropionate (3-OHP) CO2 fixation bi-cycle that has environmental sampling, in situ microenvironmental been described for these organisms (Strauss and analysis, RNA extraction and cDNA synthesis have Fuchs, 1993; Zarzycki et al., 2009) can also operate been previously described (Liu et al., 2011, 2012). mixotrophically. During mixotrophic growth, organ- Additional details of these methods are described isms simultaneously incorporate both inorganic in the Supplementary Information. All transcript carbon and organic carbon sources, such as acetate sequences from this study have been deposited in and glycolate (Zarzycki and Fuchs, 2011; Bryant the Sequence Read Archive under the accession et al., 2012). number SRP018579. Recent metagenomic characterizations of photo- trophic microbial mat communities in Octopus Spring and Mushroom Spring have revealed three Alignment and statistical analyses of cDNA sequences additional and abundant potentially photo- The sequences from the SOLiD 3.5 analysis were heterotrophic populations: ‘Candidatus Chloracido- aligned to an expanded metagenomic scaffold bacterium thermophilum’ (Kingdom Acidobacteria; database (including scaffolds that were 45kb in Bryant et al., 2007, Garcia Costas et al., 2012), size; see Supplementary Information) using the ‘Candidatus Thermochlorobacter aerophilum’ BWA (Burrows–Wheeler Alignment) algorithm (Kingdom Chlorobi; Klatt et al., 2011, Liu et al. (Li and Durbin, 2009) as previously described 2011, 2012) and a novel clade of organisms related (Liu et al., 2011). Briefly, for a sequence to be to Chloroflexi of the Class Anaerolineae (Klatt counted as mapping to a particular gene, the entire et al., 2011). These organisms are known or 50 bp sequence had to be fully aligned to the predicted to be photoheterotrophs and probably reference and at least half of the sequence had to utilizeandcompeteforsomeofthesameresources be aligned to the coding region of the gene with a as other FAPs. The abundance of these organic maximum of five mismatches