The Nitroplast and Its Relatives Support a Universal Model of Features Predicting Gene Retention in Endosymbiont and Organelle Genomes

GBE The Nitroplast and Its Relatives Support a Universal Model of Features Predicting Gene Retention in Endosymbiont and Organelle Genomes Iain G. Johnston 1,2, * Department of Mathematics, University of Bergen, Bergen, Norway 2 Computational Biology Unit, University of Bergen, Bergen, Norway *Corresponding author: E-mail: . Accepted: June 14, 2024 Abstract Endosymbiotic relationships have shaped eukaryotic life. As endosymbionts coevolve with their host, toward full integration as organelles, their genomes tend to shrink, with genes being completely lost or transferred to the host nucleus. Modern endo symbionts and organelles show diverse patterns of gene retention, and why some genes and not others are retained in these genomes is not fully understood. Recent bioinformatic study has explored hypothesized influences on these evolutionary pro cesses, finding that hydrophobicity and amino acid chemistry predict patterns of gene retention, both in organelles across eu karyotes and in less mature endosymbiotic relationships. The exciting ongoing elucidation of endosymbiotic relationships affords an independent set of instances to test this theory. Here, we compare the properties of retained genes in the nitroplast, recently reported to be an integrated organelle, two related cyanobacterial endosymbionts that form “spheroid bodies” in their host cells, and a range of other endosymbionts, with free-living relatives of each. We find that in each case, the symbiont’s genome encodes proteins with higher hydrophobicity and lower amino pKa than their free-living relative, supporting the dataderived model predicting the retention propensity of genes across endosymbiont and organelle genomes. Key words: endosymbionts, organelles, genome evolution, genome erosion. Significance As endosymbionts evolve and become more and more linked with their hosts, their genomes are often “eroded,” with many of their original genes being lost. Why some genes are lost and some retained—even as these endosymbionts be come integrated organelles—is still debated. In this note, we use data from recently reported organelles and endosym bionts to support a theory describing how properties of genes make them more or less likely to be retained through this erosion process. Introduction Eukaryotic life has numerous independent examples of endosymbiotic relationships. These include integrated or ganelles like the mitochondrion and plastid acquired bil lions of years ago (Smith and Keeling 2015), through acquisition of a cyanobacterium around 100 million years ago to form the chromatophore in Paulinella algae (Gabr et al. 2020), to more recent acquisitions of bacterial endosymbionts in insects (Husnik and Keeling 2019). Other examples include the nitrogen-fixing endosymbiont in Azolla water ferns (Peters and Meeks 1989; Ran et al. 2010), a cyanobacterial symbiont of diatoms (Flores et al. 2022), a denitrifying endosymbiont in a ciliate host (Graf et al. 2021), “spheroid body” compartments in diatoms (Nakayama et al. 2011), and a nitrogen-fixing symbiont ac companying a picoeukaryotic alga (Thompson et al. 2012), © The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Genome Biol. Evol. 16(7) https://doi.org/10.1093/gbe/evae132 Advance Access publication 20 June 2024 1 1 GBE Johnston Results Here, we analyzed a collection of pairs of symbionts and free-living partners, including the nitroplast, spheroid body endosymbionts, and several other symbionts not explored in Giannakis et al. (2022). All organelles and symbionts new ly considered showed substantial increased hydrophobicity compared with their free-living relatives (Fig. 1b). The spher oid bodies and Richelia showed a hydrophobicity increase on a similar scale to that seen in the Paulinella chromato phore (Fig. 1a). The increase was slightly greater in the nitro plast, on a similar scale to the nitrogen-fixing Nostoc azollae symbiont in the Azolla water fern (Fig. 1a). Amino pKa values were found to predict gene retention patterns in mitochondria and chloroplasts, but were not ex plicitly examined previously in other endosymbionts in Giannakis et al. (2022). Figure 1c shows the trends across the relationships explored in that study. With two excep tions (Azolla and Fokinia), amino pKa values are lower (sometimes dramatically so) in endosymbionts than in freeliving relatives, matching the behavior expected from the universal model. Plastids also show this behavior; the Plasmodium mitochondrion we consider instead has a high er average amino pKa. This is not inconsistent with the uni versal model picture: the very high difference in hydrophobicity in the Plasmodium mitochondria overcomes the pKa term in the predictive model, so that the three genes are predicted to have a high retention index. In the set of newly considered relationships in this study (nitroplasts, spheroid bodies, and others), each endosymbiont (except Wolbachia, in the same family as Fokinia) also showed lower amino pKa values than its free-living relative (Fig. 1d), again on a similar scale to the chromatophore, with this effect stronger for the nitroplast than for the spheroid bodies. The gene-by-gene correlation across our data set of hydro phobicity and amino pKa value is weak (r2 = 0.022), suggest ing that Fig. 1a–d is not just reporting the same effect twice over; the behavior in hydrophobicity is largely independent on the behavior in pKa. This reflects the fact that in the original model selection process for organelle gene retention, the two features were selected together, suggesting that they provide independent information about gene retention propensity. Significance testing for the individual comparisons in Fig. 1 is not directly meaningful, as the full sets of genes from each organism are being reported—there is no sam pling noise to account for, so statements about mean dif ferences are not subject to meaningful uncertainty. The more interesting hypothesis test relates to the observation of partnership comparisons, against the null hypothesis that hydrophobicity and pKa do not differ between sym bionts and relatives. If our symbiont–relative pairs are trea ted as independent, the probability of at least 13/14 new observations (7 partnerships, for hydrophobicity and pKa, with Wolbachia pKa disagreeing with prediction) agreeing 2 Genome Biol. Evol. 16(7) https://doi.org/10.1093/gbe/evae132 Advance Access publication 20 June 2024 which has since been characterized as an integrated organ elle dubbed the “nitroplast” (Coale et al. 2024). In each of these cases, the proto-endosymbiont originally possessed a full genome. A (...truncated)