AUTHOR=Wencker Freya D. R. , Lyon Seth E. , Breaker Ronald R. 

TITLE=Improved methods for genetic manipulation of the alkaliphile Halalkalibacterium halodurans

JOURNAL=Frontiers in Microbiology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1465811

DOI=10.3389/fmicb.2024.1465811

ISSN=1664-302X

ABSTRACT=<p>An improved approach was developed for the genetic manipulation of the Gram-positive extremophile <italic>Halalkalibacterium halodurans</italic> (formerly called <italic>Bacillus halodurans</italic>). We describe an allelic replacement method originally developed for <italic>Staphylococcus aureus</italic> that allows the deletion, mutation, or insertion of genes without leaving markers or other genetic scars. In addition, a protocol for rapid <italic>in vitro</italic> plasmid methylation and transformation is presented. The combined methods allow the routine genetic manipulation of <italic>H. halodurans</italic> from initial transformation to the desired strain in 8 days. These methods improve <italic>H. halodurans</italic> as a model organism for the study of extremophiles.</p>