# Diversity of enzymes for exopolysaccharide synthesis in the fructophilic honeybee symbiont Apilactobacillus kunkeei

## TL;DR

This study explores the evolution of enzymes in Apilactobacillus kunkeei, a honeybee symbiont, that help produce exopolysaccharides, revealing unique genetic diversity and recombination patterns.

## Contribution

The study reveals how GH70 and GH32 enzyme genes in A. kunkeei have evolved through gene duplication, deletion, and recombination, contributing to strain-specific diversity.

## Key findings

- GH70 and GH32 enzyme genes in A. kunkeei show high strain-specific diversity due to gene turnover and recombination.
- Genes encoding these enzymes evolve faster than core genes and are less affected by homologous recombination.
- Phylogroups of A. kunkeei strains reflect recombination impacts on the core genome rather than enzyme gene clusters.

## Abstract

Apilactobacillus kunkeei is a fructophilic lactic acid bacterium adapted to honeybees, their food sources and products. These bacteria synthesize exopolysaccharides thought to promote host colonization and protection against toxic compounds and stressful conditions. Homopolysaccharides consisting of glucose residues are synthesized by enzymes in the glycoside hydrolase family 70 (GH70), whereas polysaccharides that contain fructose are synthesized by family 32 (GH32) enzymes. However, the mechanisms whereby these enzymes diversify are not well understood. Here, we used a comparative genomics approach to investigate the evolution of GH70 and GH32 enzymes in the A. kunkeei population.

Based on phylogenetic inferences, the GH70 proteins in 38 reference A. kunkeei strains were sorted into glucan-binding enzymes, which were predicted to have glucansucrase and branching sucrase activities, and non-glucan binding enzymes of unknown enzymatic functions. Genes for the glucan sucrases and the branching sucrases are clustered in a chromosomal segment that also contains genes for GH32 enzymes. The number and combination of genes for the glucan-binding GH70 enzymes were mostly strain-specific, indicative of high rates of gene turnover. Neighboring genes often displayed a dramatic variability in synonymous and nonsynonymous substitution frequencies and have only rarely co-diverged. We identified short recombination tracts and a few long tracts that spanned across the cluster of genes for GH70 and GH32 enzymes. Genes encoding GH70 and GH32 enzymes evolve faster than genes encoding core proteins. The ratios of the relative effect of recombination to mutation for the core genome were estimated to 1.6 to 5.2 for A. kunkeei strains assigned to phylogroups A and B-C, respectively.

Our results suggest genes for GH32 and GH70 proteins have a unique evolutionary history in each A. kunkeei strain and have diverged by duplications, deletions, fusions, recombination events and nucleotide substitutions. We suggest that genes for GH70 enzymes have escaped the homogenizing effects of homologous recombination to a greater extent than the core genes due to rampant gene gain and loss. The results imply that the clustering of the A. kunkeei-related strains into phylogroups mostly reflects the impact of homologous recombination on the core genome.

The online version contains supplementary material available at 10.1186/s12866-025-04680-3.