Genome-wide fitness analysis of <i>Salmonella enterica</i> reveals <i>aroA</i> mutants are attenuated due to iron restriction <i>in vitro</i>

<i>Salmonella enterica</i> is a globally disseminated pathogen that is the cause of over 100 million infections per year. The resulting diseases are dependent upon host susceptibility and the infecting serovar. As <i>S. enterica</i> serovar Typhimurium induces a typhoid-like disease in mice, this model has been used extensively to illuminate various aspects of <i>Salmonella</i> infection and host responses. Due to the severity of infection in this model, researchers often use strains of mice resistant to infection or attenuated <i>Salmonella</i>. Despite decades of research, many aspects of <i>Salmonella</i> infection and fundamental biology remain poorly understood. Here, we use a transposon insertion sequencing technique to interrogate the essential genomes of widely used isogenic wild-type and attenuated <i>S</i>. Typhimurium strains. We reveal differential essential pathways between strains <i>in vitro</i> and provide a direct link between iron starvation, DNA synthesis, and bacterial membrane integrity.IMPORTANCE<i>Salmonella enterica</i> is an important clinical pathogen that causes a high number of deaths and is increasingly resistant to antibiotics. Importantly, <i>S. enterica</i> is used widely as a model to understand host responses to infection. Understanding how Salmonella survives <i>in vivo</i> is important for the design of new vaccines to combat this pathogen. Live attenuated vaccines have been used clinically for decades. A widely used mutation, <i>aroA</i>, is thought to attenuate <i>Salmonella</i> by restricting the ability of the bacterium to access particular amino acids. Here we show that this mutation limits the ability of <i>Salmonella</i> to acquire iron. These observations have implications for the interpretation of many previous studies and for the use of <i>aroA</i> in vaccine development.