Conserved Bacterial Genomes from Two Geographically Isolated Peritidal Stromatolite Formations Shed Light on Potential Functional Guilds
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Environmental Microbiology Reports (2021) 13(2), 126–137 doi:10.1111/1758-2229.12916 Brief Report Conserved bacterial genomes from two geographically isolated peritidal stromatolite formations shed light on potential functional guilds Samantha C. Waterworth,1 Eric W. Isemonger,2 oldest fossils of living organisms on Earth (Dupraz Evan R. Rees,1 Rosemary A. Dorrington2 and et al., 2009; Nutman et al., 2016, 2019). The study of Jason C. Kwan 1* extant stromatolite analogues may help to elucidate the 1Division of Pharmaceutical Sciences, University of biological mechanisms that led to the formation and evo- Wisconsin, Madison, WI, 53705. lution of their ancient ancestors. The biogenicity of stro- 2Department of Biochemistry and Microbiology, Rhodes matolites has been studied extensively in the hypersaline University, Grahamstown, South Africa. and marine formations of Shark Bay, Australia, and Exuma Cay, The Bahamas, respectively (Khodadad and Foster, 2012; Mobberley et al., 2015; Centeno Summary et al., 2016; Gleeson et al., 2016; Ruvindy et al., 2016; Stromatolites are complex microbial mats that form Warden et al., 2016; White et al., 2016; Babilonia lithified layers. Fossilized stromatolites are the oldest et al., 2018; Wong et al., 2018; Chen et al., 2020). The evidence of cellular life on Earth, dating back over presence of Archaea has been noted in several microbial 3.4 billion years. Modern stromatolites are relatively mats and stromatolite systems (Casaburi et al., 2016; rare but may provide clues about the function and Balci et al., 2018; Medina-Chávez et al., 2019; Chen evolution of their ancient counterparts. In this study, et al., 2020), particularly in the stromatolites of Shark we focus on peritidal stromatolites occurring at Cape Bay, where they are hypothesized to potentially fulfil the Recife and Schoenmakerskop on the southeastern role of nitrifiers and hydrogenotrophic methanogens South African coastline, the former being morpholog- (Wong et al., 2017). Studies of freshwater microbialites in ically and structurally similar to fossilized phosphatic Mexico found that there were significantly more genes stromatolites formations. Using assembled shotgun associated with phosphate uptake and metabolism than metagenomic analysis, we obtained 183 genomic bins, in other communities associated with fresh or marine of which the most dominant taxa were from the Cya- waters (Breitbart et al., 2009) and that nitrogen fixation nobacteria phylum. We identified functional gene sets by heterocystous cyanobacteria, sulfur-reducing bacteria, in genomic bins conserved across two geographically and purple sulfur bacteria was important to the formation isolated stromatolite formations, which included rela- of these particular stromatolites (Falcón et al., 2007). tively high copy numbers of genes involved in the In the current study, we sought to investigate the reduction of nitrates and phosphatic compounds. metagenomes, at a genome-resolved level, of two geo- Additionally, we found little evidence of Archaeal spe- graphically isolated (~2.8 km apart), peritidal, stromatolite cies in these stromatolites, suggesting that they may formations at Cape Recife and Schoenmakerskop, on the not play an important role in peritidal stromatolite for- eastern coast of South Africa. These formations are mor- mations, as proposed for hypersaline formations. phologically and structurally similar to fossilized phos- phatic stromatolites dating back to the Great Introduction Oxygenation Event during the Paleoproterozoic era (Büttner et al., 2020). The stromatolite formations at Cape Stromatolites are organo-sedimentary structures that Recife and Schoenmakerskop have been extensively date back between 3.4 and 3.8 billion years, forming the characterized with respect to their physical structure, nutrient, and chemical environment (Perissinotto et al., 2014; Rishworth et al., 2016, 2017b, 2019; Dodd Received 1 April, 2020; accepted 6 December, 2020. *For corre- spondence. E-mail [email protected]; Tel. (+608) 262 3829; et al., 2018) and they experience regular shifts in salinity Fax +1 608 2625 345. due to tidal overtopping and groundwater seepage © 2020 Society for Applied Microbiology and John Wiley & Sons Ltd Functional bacterial guilds in stromatolites 127 (Rishworth et al., 2019). Stromatolites are impacted by (Supporting Information Fig. S1), but that all communities fluctuating environmental pressures caused by periodic were dominated by Cyanobacteria with lesser, but nota- inundation by seawater, which affects the nutrient con- ble abundances of Bacteroidetes, Alphaproteobacteria, centrations, temperature and chemistry of the system and Gammaproteobacteria (Fig. 2A). Shotgun meta- (Rishworth et al., 2016). These formations are character- genomic sequencing was performed for all 10 sites ized by their proximity to the ocean, where stromatolites (BioProject PRJNA612530), and binning of the meta- in the upper formations receive freshwater from the inflow genomic data resulted in a total of 183 bacterial genome seeps, middle formation stromatolites withstand a mix of bins (Supporting Information Table S1; Please refer to freshwater seepage and marine over-topping, and lower the Supporting Information Materials and Methods for formations are in closest contact with the ocean additional details and find all scripts used here: https:// (Perissinotto et al., 2014). The stromatolite formations at github.com/samche42/Strom_Brief_report). The phyloge- Cape Recife and Schoenmakerskop are exposed to both netic distribution and relative abundance thereof were fresh and marine water that has little dissolved inorganic approximately congruent with the 16S rRNA community phosphate and decreasing levels of dissolved inorganic profiles of each site, where Cyanobacteria were consis- nitrogen (Cape Recife: 82–9 μM, Schoenmakerskop: tently dominant (36%–89%) while Alphaproteobacteria, 424–14 μM) moving from freshwater to marine influenced Gammaproteobacteria, and Bacteroidia were less abun- formations (Rishworth et al., 2017a). Microbial communi- dant but notable bacterial classes (Fig. 2B). Furthermore, ties within these levels therefore likely experience distinct scrutiny of all contigs from Cape Recife and Scho- environmental pressures, including fluctuations in salinity enmakerskop showed that none of the datasets com- and dissolved oxygen (Rishworth et al., 2016). While car- prised more than 1.5% archaeal genes. Two low-quality bon predominantly enters these systems through archaeal bins were retrieved from each of the lower for- cyanobacterial carbon fixation, it is unclear how other mations and were classified as Woesearchaeales (order) members of the stromatolite-associated bacterial consor- and Nitrosoarchaeum (genus), respectively. The cover- tia influence mineral stratification resulting from the age of these genomes is among the lowest in each of the cycling of essential nutrients such as nitrogen, phospho- lower formation samples (Supporting Information rus, and sulfur. Table S1) and would suggest a low numerical abundance However, not much is known about the microbial of these Archaea, and it is likely that this kingdom is less assemblage of these peritidal formations. It is hypothe- important in the stromatolites found at Cape Recife and sized that the growth of peritidal stromatolites at these Schoenmakerskop. These results are largely in agree- sites is promoted by decreased levels of wave action, ment with similar studies investigating freshwater higher water alkalinity, decreased levels of salinity, and microbialites from various regions of Mexico, and fresh- decreased calcite and aragonite saturation (Dodd water microbialites in Clinton Creek, Canada, which were et al., 2018). Here, we investigate the metabolic potential dominated by Cyanobacteria and Proteobacteria but of the microbes associated with these stromatolites and included less than 2% archaeal sequence (Breitbart their potential role in stromatolite formation. et al., 2009; Centeno et al., 2012; White et al., 2015; Yanez-Montalvo et al., 2020) and is in contrast to com- munities present in hypersaline stromatolites, where Results and discussion Archaea comprise a large proportion of the microbial Stromatolite formations were classified according to their community (Wong et al., 2017, 2018). To identify bins tidal proximity as defined in previous studies, as ‘Upper’ common to both Cape Recife and Schoenmakerskop, we (freshwater inflow), ‘Middle’, and ‘Lower’ (marine- calculated pairwise average nucleotide identity (ANI) influenced) (Perissinotto et al., 2014; Rishworth between all binned genomes using ANIcalculator et al., 2016, 2017b). Samples for this study were col- (Varghese et al., 2015) and defined conserved bins as lected at low tide from the upper stromatolites at Cape sharing more than 97% ANI in two or more of the sam- Recife and Schoenmakerskop in January and April 2018 pled regions (Supporting Information Table S2; please for comparisons over time and geographic space. Addi- refer to the Supporting Information Materials and tional samples were collected in April 2018 from middle Methods for additional details and find all scripts used and lower formations for extended comparison across here: https://github.com/samche42/Strom_Brief_report). the two sites (Fig. 1). Bins classified within the Acaryochloris and Hydrococcus Preliminary assessment of the structure of bacterial genera were conserved across upper formations, and communities in these samples using 16S rRNA amplicon seven bins were