Skip to main content

METHODS article

Front. Microbiol.
Sec. Extreme Microbiology
Volume 15 - 2024 | doi: 10.3389/fmicb.2024.1465811
This article is part of the Research Topic Microbial Ecology and Biotechnological Potential of Alkaline Environments View all articles

Improved methods for genetic manipulation of the alkaliphile Halalkalibacterium halodurans

Provisionally accepted
Freya D. Wencker Freya D. Wencker 1Seth E. Lyon Seth E. Lyon 2Ronald R. Breaker Ronald R. Breaker 1,2,3*
  • 1 Howard Hughes Medical Institute, Yale University, New Haven, CT, United States
  • 2 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, United States
  • 3 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, United States

The final, formatted version of the article will be published soon.

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

    Keywords: Allelic replacement, anhydrotetracycline (ATc), Double cross-over, HaeIII methyltransferase, Homologous Recombination, inducible counter-selection, secY antisense RNA

    Received: 16 Jul 2024; Accepted: 03 Sep 2024.

    Copyright: © 2024 Wencker, Lyon and Breaker. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Ronald R. Breaker, Howard Hughes Medical Institute, Yale University, New Haven, CT, United States

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.