A Semi-Quantitative, Synteny-Based Method to Improve Functional Predictions for Hypothetical and Poorly Annotated Bacterial and Archaeal Genes

Abstract

During microbial evolution, genome rearrangement increases with increasing sequence divergence. If the relationship between synteny and sequence divergence can be modeled, gene clusters in genomes of distantly related organisms exhibiting anomalous synteny can be identified and used to infer functional conservation. We applied the phylogenetic pairwise comparison method to establish and model a strong correlation between synteny and sequence divergence in all 634 available Archaeal and Bacterial genomes from the NCBI database and four newly assembled genomes of uncultivated Archaea from an acid mine drainage (AMD) community. In parallel, we established and modeled the trend between synteny and functional relatedness in the 118 genomes available in the STRING database. By combining these models, we developed a gene functional annotation method that weights evolutionary distance to estimate the probability of functional associations of syntenous proteins between genome pairs. The method was applied to the hypothetical proteins and poorly annotated genes in newly assembled acid mine drainage Archaeal genomes to add or improve gene annotations. This is the first method to assign possible functions to poorly annotated genes through quantification of the probability of gene functional relationships based on synteny at a significant evolutionary distance, and has the potential for broad application.

Author Summary

Based on trends between gene sequence divergence and gene order divergence over time, we developed a new synteny-based method to refine functional annotation. This method uses these trends to determine the probability that any two syntenous genes (genes that are sequential in two organisms) are functionally related. Organisms that are distant relatives have few syntenous genes, but these syntenous genes have a very high probability of functional relatedness. We applied this method to newly assembled genomes of co-occurring, uncultivated acid mine drainage Archaea in order to improve their gene annotations. This application revealed important physiological differences between the co-occurring organisms in this clade, including the ability of some but not all of the Archaea to manufacture vitamin B12 and to carry out anaerobic energy metabolism. We also used this method to identify new genes possibly involved in vitamin B12 synthesis, ether lipid synthesis, molybdopterin synthesis and utilization, and microbial immunity through the CRISPR system.