Zinc acquisition mechanisms differ between environmental and virulent Francisella species

Zinc is an essential nutrient for bacterial growth. Because host cells can restrict pathogen access to zinc as an antimicrobial defense mechanism, intracellular pathogens such as Francisella must sense their environment and acquire zinc in response. In many bacteria, the conserved transcription factor Zur is a key regulator of zinc acquisition. To identify mechanisms of zinc uptake in Francisella novicida U112, RNA sequencing was performed on wild-type and putative zur mutant bacteria. Only three genes were confirmed by quantitative RT-PCR as directly regulated by Zur and zinc limitation. One of these genes, FTN_0879, is predicted to encode a protein with similarity to the zupT family of zinc transporters, which are not typically regulated by Zur. While a putative znuACB operon encoding a high affinity zinc transporter was identified in U112, expression of this operon was not controlled by Zur or zinc concentration. Disruption of zupT but not znuA in U112 impaired growth under zinc limitation, suggesting that ZupT is the primary mechanism for zinc acquisition in these conditions. In the virulent Francisella tularensis subsp. tularensis Schu S4 strain, zupT is a pseudogene and attempts to delete znuA were unsuccessful, suggesting that it is essential in this strain. A reverse TetR repression system was utilized to knockdown expression of znuA in Schu S4, revealing that znuA is required for growth under zinc limitation and contributes to intracellular growth within macrophages. Overall, this work identifies genes necessary for adapting to zinc limitation and highlights nutritional differences between environmental and virulent Francisellastrains.ImportanceFrancisella tularensis is a Tier 1 Select Agent with a high potential for lethality and no approved vaccine. A better understanding of Francisella virulence factors is required for the development of therapeutics. While acquisition of zinc has been shown to be required for virulence of numerous intracellular pathogens, zinc uptake has not been characterized in Francisella This work characterizes the Zur regulon in F. novicida and identifies two transporters that contribute to bacterial growth under zinc limitation. In addition, these data identify differences in mechanisms of zinc uptake and tolerance to zinc limitation between F. tularensis and F. novicida, highlighting the role of znuA for growth under zinc limitation in Schu S4.