ClpP2 proteasomes and SpxA1 determine Listeria monocytogenes tartrolon B hyper-resistance

The natural reservoir of the foodborne bacterium Listeria monocytogenes is the soil. From there the bacterium gains access to other environmental and animal niches and transmission to humans then occurs through consumption of contaminated plant and animal-based food. We have shown previously that L. monocytogenes uses ATP-binding cassette multi drug resistance transporters to resist antimicrobial compounds produced by competitors co-residing in its natural reservoirs. Among these transporters, the TimAB transporter conferred resistance of L. monocytogenes against tartrolon B, a boron containing macrodiolide produced by Sorangium and Teredinibacter species. Tartrolon B acts as a potassium ionophor and is sensed by the transcriptional repressor TimR, which reliefs repression of the timABR operon upon compound contact. We have isolated tartrolon B resistant suppressor mutants outside the tim locus. Surprisingly, these mutations preferentially inactivated core and accessory proteasomal genes including clpP2, which encodes the main proteolytic component of house-keeping Clp proteases. Deletion of clpP2 impaired growth and virulence but caused tartrolon B hyper-resistance. This phenotype was timAB-dependent, but neither production nor degradation of TimAB was affected in the absence of ClpP2. Combinatorial deletions of the genes encoding the three Clp ATPases showed that ClpCP2 and ClpXP2 proteasomes jointly promote tartrolon B hyper-resistance. Further genetic experiments then identified the ClpP2 substrate and transcription factor SpxA1 as another tartrolon B resistance determinant. Accumulation or overexpression of SpxA1 promoted tartrolon B resistance in a timAB-dependent manner. Our work demonstrates a novel role for the redox-responsive transcription factor SpxA1 in the resistance against a potassium ionophor and helps to better understand the genetic basis of the manifold ecological interactions of an important human pathogen in its natural ecologic niches.