# Genome assembly and protein structure modeling reveal key molecular features of divergent wmk homologs in Wolbachia

## TL;DR

The study explores the wmk gene in Wolbachia bacteria, revealing new structural features that may explain male-killing behavior and its evolution.

## Contribution

A novel wmk homolog was identified and structurally characterized, revealing a new modular architecture and functional insights.

## Key findings

- The wmk-encoded protein has a modular structure with two HTH domains and an accessory domain.
- Divergent wmk homologs differ in sequence, structure, and phylogeny from canonical variants.
- Structural features distinguish wmk from other HTH-type regulators, supporting its functional relevance.

## Abstract

Wolbachia induces female-biased sex ratios in host populations through male-specific killing, thereby enhancing its spread via maternal transmission. The prophage-associated gene wmk has been proposed as a key effector underlying this male-killing (MK) phenotype. Interestingly, wmk homologs are found across diverse Wolbachia strains, regardless of the presence of male killing, and show extensive sequence divergence. However, the functional implications of this sequence variation—particularly among distant homologs—remain poorly understood. Here, we analyzed wmk homologs from 18 Wolbachia genomes, including 17 publicly available genomes and one de novo assembled genome from the parthenium beetle. We identified a highly divergent wmk homolog in the latter and predicted its protein structure using AlphaFold2, followed by molecular dynamics simulations to characterize its molecular features. Our analyses reveal that wmk-encoded protein exhibits a modular architecture comprising two helix-turn-helix (HTH) domains and an additional accessory domain not previously described. Modeling of inter-domain interactions further supports the functional relevance of this modular organization. Comparative analyses across all homologs distinguish divergent wmk from other canonical variants based on sequence composition, structural organization, and phylogenetic clustering. Notably, the structural features that differentiate these homologs also distinguish wmk from other known HTH-type regulators. Together, our findings provide new molecular insights into the architecture and evolution of wmk, offering a framework to understand the mechanistic basis of the MK phenotype in Wolbachia.

Wolbachia-induced male killing presents a promising strategy for the biocontrol of vector and pest populations. The wmk gene has been identified as a candidate underlying this phenotype. Yet, the significance of its sequence variation—particularly between highly divergent homologs—remains unclear. Here, we characterize a divergent wmk homolog from a novel Wolbachia strain. Then, we compare sequence and structural features of wmk homologs across a total of 18 Wolbachia strains using AlphaFold2 and molecular dynamics simulations. Our results highlight key molecular features in divergent variants and provide new insights into wmk evolution, laying a basis for exploring its functional diversity across Wolbachia lineages.