The Zetaproteobacteria are a candidate class of marine iron-oxidizing bacteria that are typically found in high iron environments such as hydrothermal vent sites. As much remains unknown about these organisms due to difficulties in cultivation, single-cell genomics was used to learn more about this elusive group at Loihi Seamount. Comparative genomics of 23 phylogenetically diverse single amplified genomes (SAGs) and two isolates indicate niche specialization among the Zetaproteobacteria may be largely due to oxygen tolerance and nitrogen transformation capabilities. Only Form II ribulose 1,5-bisphosphate carboxylase (RubisCO) genes were found in the SAGs, suggesting that some of the uncultivated Zetaproteobacteria may be adapted to low oxygen and/or high carbon dioxide concentrations. There is also genomic evidence of oxygen-tolerant cytochrome c oxidases and oxidative stress-related genes, indicating that others may be exposed to higher oxygen conditions. The Zetaproteobacteria also have the genomic potential for acquiring nitrogen from numerous sources including ammonium, nitrate, organic compounds, and nitrogen gas. Two types of molybdopterin oxidoreductase genes were found in the SAGs, indicating that those found in the isolates, thought to be involved in iron oxidation, are not consistent among all the Zetaproteobacteria. However, a novel cluster of redox-related genes was found to be conserved in 10 SAGs as well as in the isolates warranting further investigation. These results were used to isolate a novel iron-oxidizing Zetaproteobacteria. Physiological studies and genomic analysis of this isolate were able to support many of the findings from SAG analyses demonstrating the value of these data for designing future enrichment strategies.

A delicate relationship exists between reef-building corals and their photosynthetic endosymbionts. Unfortunately, this relationship can be disrupted, with corals expelling these algae when temperatures rise even marginally above the average summer maximum. Interestingly, several studies indicate that failure of corals to regulate symbiont cell divisions at high temperatures may underlie this disruption; increased proliferation of symbionts may stress host cells by over-production of reactive oxygen species or by disrupting the flow of nutrients. This needs to be further investigated, so to begin deciphering the molecular mechanisms controlling the cell cycle in these organisms, we used a computational approach to identify putative cell cycle-regulating genes in the genome of the dinoflagellate Breviolum minutum. This species is important as an endosymbiont of Aiptasia pallida—an anemone that is used as a model for studying coral biology. We then correlated expression of these putative cell cycle genes with cell cycle phase in diurnally growing B. minutum in culture. This approach allowed us to identify a cyclin/cyclin-dependent kinase pair that may function in the G1/S transition—a likely point for coral cells to exert control over algal cell divisions.

Multiple mutations often have non-additive (epistatic) phenotypic effects. Epistasis is of fundamental biological relevance but is not well understood mechanistically. Adaptive evolution, i.e., the evolution of new biochemical activities, is rich in epistatic interactions. To better understand the principles underlying epistasis during genetic adaptation, we studied the evolution of TEM-1 β-lactamase variants exhibiting cefotaxime resistance. We report the collection of a library of 487 observed evolutionary trajectories for TEM-1 and determine the epistasis status based on cefotaxime resistance phenotype for 206 combinations of 2–3 TEM-1 mutations involving 17 positions under adaptive selective pressure. Gain-of-function (GOF) mutations are gatekeepers for adaptation. To see if GOF phenotypes can be inferred based solely on sequence data, we calculated the enrichment of GOF mutations in the different categories of epistatic pairs. Our results suggest that this is possible because GOF mutations are particularly enriched in sign and reciprocal sign epistasis, which leave a major imprint on the sequence space accessible to evolution. We also used FoldX to explore the relationship between thermodynamic stability and epistasis. We found that mutations in observed evolutionary trajectories tend to destabilize the folded structure of the protein, albeit their cumulative effects are consistently below the protein’s free energy of folding. The destabilizing effect is stronger for epistatic pairs, suggesting that modest or local alterations in folding stability can modulate catalysis. Finally, we report a significant relationship between epistasis and the degree to which two protein positions are structurally and dynamically coupled, even in the absence of ligand.

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