Genetic strategies such as gene disruption and fluorescent protein tagging largely contribute to understanding the molecular mechanisms of biological functions in bacteria. However, the methods for gene replacement remain underdeveloped for the filamentous bacteria Leptothrix cholodnii SP-6. Their cell chains are encased in sheath composed of entangled nanofibrils, which may prevent the conjugation for gene transfer. Here, we describe a protocol optimized for gene disruption through gene transfer mediated by conjugation with Escherichia coli S17-1 with details on cell ratio, sheath removal, and loci validation. The obtained deletion mutants for specific genes can be used to clarify the biological functions of the proteins encoded by the target genes.

Graphical overview

Hydrogen peroxide (H₂O₂) is a toxic oxidant produced as a byproduct of several biological processes. At too high levels of hydrogen peroxide cells will experience oxidative stress, leading to a cellular response to decrease its levels and to protect the cells. Previously, methods used to study and quantify intracellular H₂O₂ have been limited by both sensitivity and specificity. However, an increasing number of genetically encoded fluorescent indicators (GEFIs) are becoming available, which can specifically detect low levels of intracellular hydrogen peroxide. In this study, we use such a biosensor designed to monitor cytosolic H₂O₂ levels in the budding yeast Saccharomyces cerevisiae during continuous cultivation and in the absence of a fluorescence microscope. The fluorescent biosensor contains a peroxiredoxin protein fused to an engineered GFP molecule expressed from a commonly used yeast plasmid (pRS416-TEF1). The peroxiredoxin-based fluorescent indicator reduces H₂O₂, ultimately resulting in a GFP signal being emitted by the sensor. Here, we apply this biosensor to study cytosolic H₂O₂ levels in S. cerevisiae strains with and without recombinant protein production.

Graphic abstract:

Schematic overview of experimental steps.