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This content will become publicly available on February 18, 2026

Title: Genomic re-sequencing reveals mutational divergence across genetically engineered strains of model archaea
ABSTRACT Archaeal molecular biology has been a topic of intense research in recent decades as their role in global ecosystems, nutrient cycles, and eukaryotic evolution comes to light. The hypersaline-adapted archaeal speciesHalobacterium salinarumandHaloferax volcaniiserve as important model organisms for understanding archaeal genomics, genetics, and biochemistry, in part because efficient tools enable genetic manipulation. As a result, the number of strains in circulation among the haloarchaeal research community has increased in recent decades. However, the degree of genetic divergence and effects on genetic integrity resulting from the creation and inter-lab transfer of novel lab stock strains remain unclear. To address this, we performed whole-genome re-sequencing on a cross-section of wild-type, parental, and knockout strains in both model species. Integrating these data with existing repositories of re-sequencing data, we identify mutations that have arisen in a collection of 60 strains, sampled from two species across eight different labs. Independent of sequencing, we construct strain lineages, identifying branch points and significant genetic events in strain history. Combining this with our sequencing data, we identify small clusters of mutations that definitively separate lab strains. Additionally, an analysis of gene knockout strains suggests that roughly one in three strains currently in use harbors second-site mutations of potential phenotypic impact. Overall, we find that divergence among lab strains is thus far minimal, though as the archaeal research community continues to grow, careful strain provenance and genomic re-sequencing are required to keep inter-lab divergence to a minimum, prevent the compounding of mutations into fully independent lineages, and maintain the current high degree of reproducible research between lab groups. IMPORTANCEArchaea are a domain of microbial life whose member species play a critical role in the global carbon cycle, climate regulation, the human microbiome, and persistence in extreme habitats. In particular, hypersaline-adapted archaea are important, genetically tractable model organisms for studying archaeal genetics, genomics, and biochemistry. As the archaeal research community grows, keeping track of the genetic integrity of strains of interest is necessary. In particular, routine genetic manipulations and the common practice of sharing strains between labs allow mutations to arise in lab stocks. If these mutations affect cellular processes, they may jeopardize the reproducibility of work between research groups and confound the results of future studies. In this work, we examine DNA sequences from 60 strains across two species of archaea. We identify shared and unique mutations occurring between and within strains. Independently, we trace the lineage of each strain, identifying which genetic manipulations lead to observed off-target mutations. While overall divergence across labs is minimal so far, our work highlights the need for labs to continue proper strain husbandry.  more » « less
Award ID(s):
1651117 1936024
PAR ID:
10616370
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ; ; ;
Editor(s):
Nikel, Pablo Ivan
Publisher / Repository:
American Society for Microbiology Press
Date Published:
Journal Name:
mSystems
Volume:
10
Issue:
2
ISSN:
2379-5077
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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