Photosynth Res DOI 10.1007/s11120-013-9890-6 REGULAR PAPER Metabolic potential of lithifying cyanobacteria-dominated thrombolitic mats Jennifer M. Mobberley • Christina L. M. Khodadad • Jamie S. Foster Received: 19 April 2013 / Accepted: 9 July 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract Thrombolites are unlaminated carbonate depos- Introduction its formed by the metabolic activities of microbial mats and can serve as potential models for understanding the molecular Microbialites are carbonate-depositing ecosystems that are mechanisms underlying the formation of lithifying commu- the products of multiple microbial metabolisms and com- nities. To assess the metabolic complexity of these ecosys- plex biogeochemical cycling. Microbialites have a long tems, high throughput DNA sequencing of a thrombolitic mat fossil record dating back billions of years and are generally metagenome was coupled with phenotypic microarray anal- thought to be one of the oldest known ecosystems on Earth ysis. Functional protein analysis of the thrombolite commu- (Grotzinger and Knoll 1999). Microbialites are distin- nity metagenome delineated several of the major metabolic guished by their carbonate macrostructure. Those micro- pathways that influence carbonate mineralization including bialites with a laminated macrostructure are referred to as cyanobacterial photosynthesis, sulfate reduction, sulfide stromatolites, whereas microbialites with unlaminated oxidation, and aerobic heterotrophy. Spatial profiling of clotted fabrics are known as thrombolites. The laminated metabolite utilization within the thrombolite-forming stromatolites are the result of iterative microbial growth microbial mats suggested that the top 5 mm contained a more that accrete through the precipitation of calcium carbonate metabolically diverse and active community than the deeper as well as the trapping and binding of inorganic sediment within the mat. This study provides evidence that despite the (Reid et al. 2000; Macintyre et al. 2000; Paerl et al. 2001; lack of mineral layering within the clotted thrombolite Andres et al. 2006). In contrast to the stromatolites, the structure there is a vertical gradient of metabolic activity thrombolites show a high degree of heterogeneity and within the thrombolitic mat community. This metagenomic variability in carbonate precipitation; however, the under- profiling also serves as a foundation for examining the active lying microbial processes that form these thrombolitic role individual functional groups of microbes play in coor- clotted structures are not well understood. dinating metabolisms that lead to mineralization. To explore this issue further, the thrombolites of High- borne Cay, an island located in the Exuma Sound, The Keywords Thrombolites Á Microbial mats Á Bahamas were targeted. At Highborne Cay, both stromat- Metagenome Á Photosynthesis Á Carbonate olites and thrombolites actively grow in the subtidal and mineralization Á Microbialites intertidal zones, respectively. The thrombolites of High- borne Cay form large, unlaminated carbonate structures ranging in size from a few centimeters to several meters Electronic supplementary material The online version of this wide (Fig. 1a). Overlaying these thrombolitic carbonate article (doi:10.1007/s11120-013-9890-6) contains supplementary material, which is available to authorized users. structures is a thick (*1 cm) microbial mat (Fig. 1b) comprised of a diverse microbial consortium, which is J. M. Mobberley Á C. L. M. Khodadad Á J. S. Foster (&) thought to drive the precipitation of extracellular carbonate Department of Microbiology and Cell Science, University of through their various metabolic activities (Planavsky et al. Florida, Space Life Sciences Lab, Kennedy Space Center, FL 32899, USA 2009; Myshrall et al. 2010; Mobberley et al. 2012). There e-mail: jfoster@ufl.edu have been several recent studies that have characterized the 123 Photosynth Res taphonomy of the thrombolite carbonate structures (Plan- The production of EPS material has been shown to be a avsky and Ginsburg 2009), the surrounding oolitic sand critical factor associate with carbonate precipitation in grains (Edgcomb et al. 2013), as well as the biogeochem- modern microbialites. The EPS matrix within microbial mats istry and microbial diversity of the thrombolite-forming not only serves as a structural support for the growing com- microbial mats, from here on referred to as thrombolitic munity (Decho 1990), but it can also chelate cations, such as mats (Desnues et al. 2008; Myshrall et al. 2010; Mobberley Ca2? and function as nucleation sites for calcium carbonate et al. 2012). These previous analyses have revealed several nucleation (Decho 2000; Dupraz and Visscher 2005). The key functional groups of organisms associated with the EPS associated with the adjacent stromatolites of Highborne mats and include: phototrophs, aerobic heterotrophic bac- Cay are predominantly generated by cyanobacteria and sul- teria, sulfate-reducing bacteria, sulfide-oxidizing bacteria, fate-reducing bacteria (Decho 2000; Braissant et al. 2007). and fermentative bacteria. All these guilds have been The cyanobacterial EPS material contains approximately shown to be critical for carbonate precipitation and/or 50 % carbohydrates, such as mannose, xylose and fucose, dissolution in other lithifying microbial mat ecosystems, whereas the remaining material is a mixture of amino acids, such as stromatolites (e.g., Dupraz and Visscher 2005; uronic acids, and glycans (Kawaguchi and Decho 2000). Visscher and Stolz 2005; Baumgartner et al. 2009). Through the heterotrophic degradation of the EPS material in Together, these groups of organisms generate steep vertical the stromatolites, the Ca2? binding capacity of the EPS can be chemical gradients within the thrombolites with large diel altered resulting in the release of Ca2? ions, which can fluctuations. For example, O2 levels throughout the diel increase the saturation index of the microenvironment and cycle can go from undetectable to several-fold supersatu- promote localized areas of carbonate precipitation within the rated in the top 1–5 mm of the lithifying mat surface mat community (Dupraz and Visscher 2005). (Myshrall et al. 2010). The dominant organisms responsi- Despite recent studies regarding the viral and microbial ble for this O2-rich layer are photosynthetic cyanobacteria, diversity, sedimentology, and biogeochemistry of the Ba- which have been shown to be a driving force in the bio- hamian thrombolitic mats (Desnues et al. 2008; Planavsky geochemical cycling in thrombolites (Myshrall et al. 2010). and Ginsburg 2009; Planavsky et al. 2009; Myshrall et al. In the thrombolitic mats of Highborne Cay, one of the 2010; and Mobberley et al. 2012; Edgcomb et al. 2013) prominent cyanobacteria that comprises the upper few there remains an overall lack of understanding of the millimeters of the thrombolitic mats are calcified filamen- molecular mechanisms that underlie and regulate the tous Dichothrix spp. (Fig. 1c; Planavsky et al. 2009; metabolisms associated with these communities. In this Mobberley et al. 2012), which are characterized by their study, we survey the metabolic potential of thrombolitic basal heterocysts and tapered apical ends (Planavsky et al. mats by sequencing the metagenome using massively 2009). These bundles of vertically orientated filaments parallel DNA sequencing. The metagenomic sequencing produce copious amounts of exopolymeric substances was coupled with phenotypic microarray analysis, which (EPS) and appear to be ‘‘hot spots’’ for carbonate precip- assessed vertical metabolite utilization patterns throughout itation in the thrombolitic mats. The carbonate deposition the thrombolitic mats, thereby generating a spatial profile is thought to be due to the elevated rates of photosynthesis, of metabolic activities. These two approaches help to which increases the pH and the carbonate saturation state increase our understanding of the underlying molecular within the mats and facilitates carbonate precipitation pathways and overall genomic complexity associated with (Dupraz and Visscher 2005). these unlaminated lithifying communities. abc 1 2 3 Fig. 1 Modern thrombolites of Highborne Cay, Bahamas. a Throm- contained the lower portion of the mat between 5 and 9 mm. bolitic build-ups along the intertidal zone. Bar = 30 cm. b Cross Bar = 1 cm. c Within the upper Zone 1 there is an increase in the section of thrombolitic microbial mat demarking three zones within relative abundance of the filamentous cyanobacterium Dichothix spp. the mat. Zone 1 comprised the upper 3 mm of the mat, whereas Zone (arrows) which appear as hot spots of carbonate deposition within the 2 consisted of 3–5 mm beneath the surface of the mat, and Zone 3 thrombolitic mats. Bar = 0.5 mm 123 Photosynth Res Materials and methods sequencing data was deposited into GenBank NCBI short read archive SRP021141. Thrombolitic mat sample collection Analysis of thrombolitic mat metagenomes Thrombolitic mats were collected from the island of Highborne Cay, The Bahamas (76°490 W, 24°430N) in Each of the three metagenomic libraries (Thr-A, Thr-B, February 2010 from an intertidal thrombolite platform and Thr-C) was analyzed and annotated with the Me- located at Site 5 (Fig. 1a; Andres and Reid 2006). Based on taGenome Rapid Annotation using Subsystem Technology previous studies there are four known types of thrombolitic (MG-RAST) version 3.3 pipeline (Meyer et al. 2008) and mats (Myshrall et al. 2010;