Work

Strain handling exposes IS-mediated recurrent genome reduction at a dynamic accessory locus in Komagataeibacter rhaeticus

Year: 2026

Type: preprint

Abstract: Bacterial genomes are dynamic and harbour accessory regions that can be gained, lost, or rearranged in response to selective pressures. Members of the genus Komagataeibacter are used for bacterial cellulose production and researched as platforms for engineered living materials. However, their genomes are rich in mobile genetic elements (MGEs), including insertion sequences (ISs), genomic islands and prophage remnants, which drive genome rearrangements under agitated cultivation conditions. Whether this genome architecture contributes to genomic changes during routine strain maintenance and distribution remains poorly understood. Comparative genome sequencing of independent Komagataeibacter rhaeticus iGEM stocks revealed recurrent loss of a 210 kbp chromosomal region flanked by homologous IS5 sequences. This deletion was identified in a stock received as an agar stab and reproduced from an intact-genome population subjected to simulated agar-stab handling. Phylogenomic analysis of the affected locus revealed gene-tree/species-tree discordance, heterogeneous GC content and multiple ISs, supporting its characterization as a dynamic accessory region. Populations carrying the larger genome reduction showed improved growth and cellulose production relative to the intact genome reference, whereas populations carrying a smaller nested deletion showed reduced performance. These findings demonstrate that routine strain handling is not necessarily genomically neutral and can expose structural genomic variabilities that generate population-level genomic heterogeneity with phenotypic consequences. For bacteria with MGE-rich genomes, handling history may represent an underappreciated source of genomic and phenotypic variation between laboratories. More broadly, identifying unstable accessory regions may highlight genomic content that is dispensable under laboratory conditions, providing candidates for genome streamlining and strain improvement. Importance Bacteria are commonly shared between laboratories in small tubes filled with agar gel, a practice known as agar stab. This study shows that this common practice can trigger the unexpected loss of a large genomic region in Komagataeibacter rhaeticus. Identifying such genome instability is important as this bacterium is increasingly used for industrial bacterial cellulose production. Bacteria carrying the larger deletion grew faster and produced more cellulose, while those with a partial deletion performed worse. Our findings highlight how bacteria shared between laboratories may carry overlooked genetic differences that can contribute to phenotypic differences among research groups and provide potential targets for future genome reduction engineering to improve growth and cellulose production.

Source: bioRxiv (Cold Spring Harbor Laboratory)

DOI: 10.64898/2026.10.08.757553