Geobiology (2012), 10, 250–267 DOI: 10.1111/j.1472-4669.2012.00322.x Cyanobacterial life at low O2: community genomics and function reveal metabolic versatility and extremely low diversity in a Great Lakes sinkhole mat A. A. VOORHIES,1 B. A. BIDDANDA,2 S. T. KENDALL,2 S. JAIN,1,3 D. N. MARCUS,1 S. C. NOLD,4 N. D. SHELDON1 ANDG.J.DICK1,3,5 1Deptartment of Earth and Environmental Sciences, University of Michigan, Ann Arbor, MI, USA 2Annis Water Resources Institute, Grand Valley State University, Muskegon, MI, USA 3Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA 4Biology Department, University of Wisconsin-Stout, Menomonie, WI, USA 5Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI, USA ABSTRACT Cyanobacteria are renowned as the mediators of Earth’s oxygenation. However, little is known about the cyano- bacterial communities that flourished under the low-O2 conditions that characterized most of their evolutionary history. Microbial mats in the submerged Middle Island Sinkhole of Lake Huron provide opportunities to investi- gate cyanobacteria under such persistent low-O2 conditions. Here, venting groundwater rich in sulfate and low in O2 supports a unique benthic ecosystem of purple-colored cyanobacterial mats. Beneath the mat is a layer of carbonate that is enriched in calcite and to a lesser extent dolomite. In situ benthic metabolism chambers revealed that the mats are net sinks for O2, suggesting primary production mechanisms other than oxygenic photosynthesis. Indeed, 14C-bicarbonate uptake studies of autotrophic production show variable contributions from oxygenic and anoxygenic photosynthesis and chemosynthesis, presumably because of supply of sulfide. These results suggest the presence of either facultatively anoxygenic cyanobacteria or a mix of oxygenic ⁄ anoxy- genic types of cyanobacteria. Shotgun metagenomic sequencing revealed a remarkably low-diversity mat com- munity dominated by just one genotype most closely related to the cyanobacterium Phormidium autumnale,for which an essentially complete genome was reconstructed. Also recovered were partial genomes from a second genotype of Phormidium and several Oscillatoria. Despite the taxonomic simplicity, diverse cyanobacterial genes putatively involved in sulfur oxidation were identified, suggesting a diversity of sulfide physiologies. The dominant Phormidium genome reflects versatile metabolism and physiology that is specialized for a communal lifestyle under fluctuating redox conditions and light availability. Overall, this study provides genomic and physi- ologic insights into low-O2 cyanobacterial mat ecosystems that played crucial geobiological roles over long stretches of Earth history. Received 26 May 2011; accepted 11 January 2012 Corresponding author: G. J. Dick. Tel.: 734 763 3228; fax: 734 763 4690; e-mail: [email protected] (GOE) (Bekker et al., 2004). Conversely, recent work also INTRODUCTION suggests that cyanobacteria capable of anoxygenic photosyn- Cyanobacteria mediated Earth’s oxygenation and thus played thesis may have subsequently perpetuated an extended low- a central role in geochemical and biological evolution. They O2 phase of Earth’s history (Johnston et al., 2009). This are widely recognized as the innovators of oxygenic photosyn- intermediate stage of Earth’s redox history lasted for at least a thesis, in which water provides electrons for photosynthesis billion years and was characterized by a low-O2 atmosphere and O2 is released as a by-product (Blankenship et al., 2007). and redox-stratified oceans, where sulfide–O2 interfaces This cyanobacterial metabolism is thought to have driven would have been prevalent (Johnston et al., 2009; Lyons a significant increase in atmospheric O2 concentration et al., 2009). Despite the large portion of cyanobacterial 2.4 billion years ago known as the great oxidation event evolution that occurred while O2 was scarce, and the critical 250 Ó 2012 Blackwell Publishing Ltd Community genomics and function of a cyanobacterial mat at low O2 251 geobiological turning points that occurred under such condi- factors that affect competition between versatile cyanobacteria tions (Falkowski et al., 2008; Johnston et al., 2009), little is and other anoxygenic bacteria. Many studies have focused on known about the genetic or physiological characteristics of cy- stratified cyanobacterial mat communities where O2 and anobacteria that thrive under persistent sulfide-rich and ⁄ or sulfide concentrations fluctuate on diel cycles; often cyano- O2-limited conditions. bacteria are exposed to sulfidic conditions at night and oxic Modern cyanobacteria exhibit a range of physiologies in the conditions during the day (Richardson & Castenholz, 1987). presence of sulfide. Most are highly sensitive to sulfide because However, few studies have focused on cyanobacteria that of irreversible blockage of the H2O-splitting component of thrive under persistent low-O2 conditions. photosystem II (Cohen et al., 1986; Miller & Bebout, 2004). Here, we investigate cyanobacterial mats inhabiting such a However, cyanobacteria inhabiting anoxic or hypoxic envi- persistently low-O2 environment, the submerged Middle ronments that are regularly exposed to sulfide have developed Island Sinkhole (MIS) in Lake Huron (Fig. 1). Groundwater strategies for sulfide tolerance and even utilization (Casten- that gently vents into the MIS bottom (water depth 23 m) holz, 1976, 1977; Garlick et al., 1977; Oren et al., 1977; has significantly different physical and chemical properties Bu¨hring et al., 2011). Cohen et al., (1986) described several than Lake Huron water, with a lower temperature (7–9 vs. adaptations to sulfide, ranging from sulfide-resistant oxygenic 4–25 °C), lower pH (7.1 vs. 8.3), lower concentrations of ) photosynthesis to the ability to utilize sulfide as the electron dissolved oxygen (0–2 vs. 5–11 mg L 1), lower oxidation– donor for anoxygenic photosynthesis. Different cyanobacteri- reduction potential ()134 vs. 500 mV), and higher specific ) al species sharing the same oxic ⁄ anoxic interfacial environ- conductivity (2.3 vs. 0.3 mS cm 1) (Biddanda et al., 2009). ment often exhibit distinct sulfide physiologies, with different The high conductivity of venting groundwater is attributable ) sulfide optima and tolerance (Garlick et al., 1977; Jorgensen to high concentrations of dissolved sulfate (1250 mg L 1), ) ) et al., 1986). Such cyanobacteria that are capable of tolerating carbonate (48 mg L 1), and chloride (25 mg L 1)ions sulfide or using it for anoxygenic photosynthesis are phyloge- derived from interactions with subsurface Devonian evapor- netically diverse and spread throughout the phylum Cyano- ites (Black, 1983; Ruberg et al., 2008). This dense ground- bacteria (Miller & Bebout, 2004). water forms a thin (1 m), visibly stratified benthic layer that Despite the phylogenetically widespread nature of anoxy- persists perennially except for brief disruptions because of genic photosynthesis among the cyanobacteria, the biochemi- major storms (Ruberg et al., 2008). The low-O2 conditions cal mechanisms of this process and its genetic underpinnings of the groundwater inhibit typical Lake Huron biological have been studied in just a few cyanobacterial strains, primarily communities, favoring purple-colored cyanobacterial mats Geitlerinema sp. PCC 9228 (formerly Oscillatoria limnetica). with finger-like protrusions (Fig. 2) that have not been found When confronted with sulfide in the presence of light, this elsewhere in the Great Lakes (Biddanda et al., 2009). Beneath organism rapidly switches from oxygenic to anoxygenic pho- the mats are stratified microbial layers of sulfide-oxidizing tosynthesis by an inducible process that requires protein bacteria, sulfate-reducing bacteria, and methanogens (Nold synthesis (Cohen et al., 1975b; Oren & Padan, 1978). et al., 2010a). Molecular diversity studies (Nold et al., Photosystem I receives electrons from sulfide and transfers 2010a) have revealed that they are dominated by cyanobacte- them to the electron transport chain to drive proton pumping ria remarkably similar (>98% 16S rRNA gene sequence iden- (Belkin & Padan, 1978), and extracellular globules of elemen- tity) to Phormidium autumnale isolates from the Arctic and tal sulfur are generated as an end-product (Cohen et al., Antarctic (Taton et al., 2006a,b; Comte et al., 2007), and 1975a). Biochemical and genetic methods have identified host to distinctive archaeal and eukaryotic communities (Nold genes encoding the enzyme that oxidizes sulfide, sulfide qui- et al., 2010b). Furthermore, carbon, nitrogen, and sulfur sta- none reductase (SQR), which transfers electrons from sulfide ble isotopic signatures of sinkhole chemistry have been to the quinone pool (Arieli et al., 1994; Schu¨tz et al., 1997; detected in the surrounding environment and food web Bronstein et al., 2000). In Geitlerinema sp. 9228, these sul- (Sanders et al., 2011). fide-derived electrons are used for photosynthesis or nitrogen Little attention has been paid to the potential for the MIS fixation, whereas in the cyanobacterium Aphanothece halophy- mats as analogs for ancient microbial mat ecosystems. Micro- tica and the yeast Schizosaccharomyces pombe, SQR is thought bial mats are widespread throughout the Precambrian rock to oxidize sulfide for the purpose of detoxification (Bronstein record, often as carbonate-associated stromatolites (Grotzin- et al., 2000). ger & Knoll, 1999). In addition to their low-O2 habitat and Investigation of modern