The environmental range exhibited by known members of the SBR1031/Aggregatilineales was previously somewhat limited. Previously recovered genomes of members of this group were sourced from hot spring environments (Ward et al., 2018a, Ward et al., 2019b), with the single known isolate isolated from subseafloor sediments (Nakahara et al. 2019). 16S rRNA sequences of SBR1031 have been recovered from more diverse environments including hot springs (Ward et al., 2017a, Ward 2017), contaminated soils (Militon et al. 2010), and wastewater (Björnsson et al. 2002). Recovery of MAGs belonging to this order from carbonate tidal flats therefore expands the available genomic diversity and known range of Aggregatilineales to environments that also have an extensive geological record.
The SBR1031 MAGs reported here encoded similar sets of functional genes to previously reported members of this order (Figure 3). Like previously described members of SBR1031/Aggegatilineales (e.g. Ward et al., 2018a), these organisms encode aerobic respiration via an A-family heme copper O2 reductase, and contain both a bc complex and an alternative complex III (Ward et al. 2018a); based on these electron transport chain complexes, it is likely that these organisms are at least facultatively aerobic. All genomes described here (except for TC_22, the least complete genome) also encode a bd oxidase (O2 reductase) capable of functioning for O2 detoxification or respiration at low O2 concentrations (Borisov et al. 2011)—a trait observed in both aerobic and anaerobic members of the Anaerolineae class (e.g. Hemp et al. 2015, Pace et al. 2015, Ward et al. 2015, Ward et al. 2018a).
Of the five SBR1031/Aggregatilineales genomes reported here, three encode partial or full components necessary for phototrophic energy transduction via a Type 2 reaction center. TC_71 and TC_152 encode complete sets of marker genes for phototrophy, including those encoding PufL and PufM subunits of the reaction center and bacteriochlorophyll synthesis (e.g. BchX, BchY, and BchZ). MetaPOAP False Positive estimates for phototrophy in these organisms were low (<0.04), suggesting that it is very unlikely that these genes were recovered as a result of contamination in the MAGs. The TC_22 genome encodes the BchXYZ complex but did not recover genes for PufL or PufM; MetaPOAP False Positive and False Negative estimates were similarly low (~0.04) for this genome, and based on this analysis it remains unclear whether or not this organism contains a complete set of genes for phototrophy. However, the gene cluster encoding BchX, BchY, and BchZ in TC_22 is located on the end of a contig, and the region of the chromosome syntenous to that encoding PufL and PufM in other phototrophic Chloroflexota (e.g. 6.5kb upstream of bchx, bchy, and bchz in TC_152) is missing in the TC_22 genome. Based on this, it is possible that this organism hosts a complete phototrophy pathway but that some genes simply were not recovered in the MAG. Like previously described phototrophs in SBR1031, these organisms do not encode a BchLNB complex or the capacity for carbon fixation via either the 3-hydroxypropionate bi-cycle or the Calvin cycle (Ward et al. 2018a). It is worth noting, however, that TC_22 does encode a Form IV rubisco-like protein on a small contig; however enzymes in this family are not capable of catalyzing CO2 fixation and instead are used for a variety of other functions (Tabita et al., 2008) and this genome does not encode phosphoribulose kinase.
Comparisons of organismal (based on concatenated ribosomal proteins and other vertically inherited markers) and phototrophy protein (e.g. PufL, PufM) phylogenies indicated substantial incongruences between organismal and phototrophy tree topologies. These relationships are indicative of a history of horizontal gene transfer (Figure 2b) (e.g. Raymond et al. 2002). In particular, the reaction centers found in members of SBR1031 branch with those of Roseiflexus rather than as a clade separate from those of Chloroflexia (e.g. Chloroflexus+Roseiflexus), suggesting that horizontal transfer of phototrophy proteins occurred from the Roseiflexus branch to members of SBR1031—a pattern previously recognized in other members of the SBR1031/Aggegatilineales (Ward et al. 2018a). Interestingly, like Roseiflexus and some other phototrophic members of SBR1031/Aggegatilineales (Ward et al. 2018a), TC_71 and TC_152 encode fused pufL/pufM genes encoding the two subunits of the Type 2 reaction center heterodimer. Together with reaction center protein phylogenic relationships, this observation indicated that the reaction centers of these organisms are more closely related to those of the Roseiflexus lineage of Chloroflexia than to the more closely related Ca. Roseilinea gracile, which encodes unfused pufL and pufM genes. A corollary of these observations of is that phototrophy in SBR1031 must postdate the acquisition and diversification of phototrophy in the Chloroflexia, events that have been estimated to have occurred in the last ~1 billion years (Shih et al. 2017).