Nikolaos Vakirlis1,2 and Anne Kupczok3 1Institute For Fundamental Biomedical Research, B.S.R.C. “Alexander Fleming,” Vari 166 72, Greece; 2Institute for General Microbiology, Kiel University, 24118 Kiel, Germany; 3Bioinformatics Group, Wageningen University, 6700 PB Wageningen, The Netherlands Corresponding authors: vakirlisfleming.gr, anne.kupczokwur.nl Abstract

Species-specific genes, also known as orphans, are ubiquitous across life's domains. In prokaryotes, species-specific orphan genes (SSOGs) are mostly thought to originate in external elements such as viruses followed by horizontal gene transfer, whereas the scenario of native origination, through rapid divergence or de novo, is mostly dismissed. However, quantitative evidence supporting either scenario is lacking. Here, we systematically analyzed genomes from 4644 human gut microbiome species and identified more than 600,000 unique SSOGs, representing an average of 2.6% of a given species’ pangenome. These sequences are mostly rare within each species yet show signs of purifying selection. Overall, SSOGs use optimal codons less frequently, and their proteins are more disordered than those of conserved genes (i.e., non-SSOGs). Importantly, across species, the GC content of SSOGs closely matches that of conserved ones. In contrast, the ∼5% of SSOGs that share similarity to known viral sequences have distinct characteristics, including lower GC content. Thus, SSOGs with similarity to viruses differ from the remaining SSOGs, contrasting an external origination scenario for most of them. By examining the orthologous genomic region in closely related species, we show that a small subset of SSOGs likely evolved natively de novo and find that these genes also differ in their properties from the remaining SSOGs. Our results challenge the notion that external elements are the dominant source of prokaryotic genetic novelty and will enable future studies into the biological role and relevance of species-specific genes in the human gut.

Received January 11, 2024. Accepted June 12, 2024.