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Brucella ovis is an ovine intracellular pathogen with tropism for the male genital tract. To establish and maintain infection, B. ovis must survive stressful conditions inside host cells, including low pH, nutrient limitation, and reactive oxygen species. The same conditions are often encountered in axenic cultures during stationary phase. Studies of stationary phase may thus inform our understanding of Brucella infection biology, yet the genes and pathways that are important in Brucella stationary-phase physiology remain poorly defined. We measured fitness of a barcoded pool of B. ovis Tn- himar mutants as a function of growth phase and identified cysE as a determinant of fitness in stationary phase. CysE catalyzes the first step in cysteine biosynthesis from serine, and we provide genetic evidence that two related enzymes, CysK1 and CysK2, function redundantly to catalyze cysteine synthesis at steps downstream of CysE. Deleting cysE (Δ cysE ) or both cysK1 and cysK2 (Δ cysK1 Δ cysK2 ) results in premature entry into stationary phase, reduced culture yield, and sensitivity to exogenous hydrogen peroxide. These phenotypes can be chemically complemented by cysteine or glutathione. Δ cysE and Δ cysK1 Δ cysK2 strains have no defect in host cell entry in vitro but have significantly diminished intracellular fitness between 2 and 24 h postinfection. Our study has uncovered unexpected redundancy at the CysK step of cysteine biosynthesis in B. ovis and demonstrates that cysteine anabolism is a determinant of peroxide stress survival and fitness in the intracellular niche.

Brucella ovis Cysteine Biosynthesis Contributes to Peroxide Stress Survival and Fitness in the Intracellular Niche