Genetic Approaches to Uncover Gene Products Involved in Iron-Sulfur Protein Maturation: High-Throughput Genomic Screening Using Transposon Sequencing

Iron-sulfur (Fe-S) clusters are one of the most ubiquitous and versatile prosthetic groups exploited by nature. Fe-S clusters aid in conducting redox reactions, carbon activation, and environmental sensing. This chapter presents an overview of the genetic approaches that have been useful for identifying and characterizing bacterial factors involved in Fe-S protein assembly. Traditional genetic screens that assess viability or conditional auxotrophies and bioinformatic approaches have identified the majority of the described genes utilized for Fe-S protein assembly. Herein, we expand upon this list of genetic methods by detailing the use of transposon sequencing (TnSeq) to identify gene products that are necessary for the proper function of metabolic pathways that require Fe-S enzymes. TnSeq utilizes the power of genomics and massively parallel DNA sequencing to allow researchers to quantify the necessity of individual gene products for a specific growth condition. This allows for the identification of gene products or gene networks that have a role in a given metabolic process but are not essential for the process. An advantage of this approach is that it allows researchers to identify mutants that have partial phenotypes that are often missed using traditional plate-based selections. Applying TnSeq to address questions of Fe-S protein maturation will result in a more comprehensive understanding of genetic interactions and factors utilized in Fe-S biogenesis and Fe-S protein assembly.