In all domains of life, Hsp70 chaperones preserve protein homeostasis by promoting protein folding and degradation and preventing protein aggregation. We now report that the Hsp70 from the bacterial pathogen Salmonella enterica serovar Typhimurium-termed DnaK-independently reduces protein synthesis in vitro and in S. Typhimurium facing cytoplasmic Mg2+ starvation, a condition encountered during infection. This reduction reflects a 3-fold increase in ribosome association with DnaK and a 30-fold decrease in ribosome association with trigger factor, the chaperone normally associated with translating ribosomes. Surprisingly, this reduction does not involve J-domain cochaperones, unlike previously known functions of DnaK. Removing the 74 C-terminal amino acids of the 638-residue long DnaK impeded DnaK association with ribosomes and reduction of protein synthesis, rendering S. Typhimurium defective in protein homeostasis during cytoplasmic Mg2+ starvation. DnaK-dependent reduction in protein synthesis is critical for survival against Mg2+ starvation because inhibiting protein synthesis in a dnaK-independent manner overcame the 10,000-fold loss in viability resulting from DnaK truncation. Our results indicate that DnaK protects bacteria from infection-relevant stresses by coordinating protein synthesis with protein folding capacity.

Molecular chaperones are critical for protein homeostasis. In bacteria, chaperone trigger factor (TF) folds proteins co-translationally, and chaperone DnaK requires a J-domain cochaperone and nucleotide exchange factor GrpE to fold proteins largely post-translationally. However, when the pathogen Salmonella enterica serovar Typhimurium faces the infection-relevant condition of cytoplasmic Mg²⁺ starvation, DnaK reduces protein synthesis independently. This raises the possibility that bacteria differentially express chaperones and cochaperones. We now report that S. Typhimurium responds to cytoplasmic Mg²⁺ starvation by increasing mRNA amounts of dnaK while decreasing those of the TF-encoding gene tig and J-domain cochaperone genes dnaJ and djlA. This differential strategy requires the master regulator of Mg²⁺ homeostasis and virulence PhoP, which increases dnaK mRNA amounts by lowering the ATP concentration, thereby hindering proteolysis of the alternative sigma factor RpoH responsible for dnaK transcription. We also establish that DnaK exerts negative feedback on the RpoH protein and RpoH-dependent transcripts independently of J-domain cochaperones. Thus, bacteria express chaperones and cochaperones coordinately or differentially depending on the specific stress perturbing protein homeostasis.IMPORTANCEMolecular chaperones typically require cochaperones to fold proteins and to prevent protein aggregation, and the corresponding genes are thus coordinately expressed. We have now identified an infection-relevant stress condition in which the genes specifying chaperone DnaK and cochaperone DnaJ are differentially expressed despite belonging to the same operon. This differential strategy requires the master regulator of Mg²⁺ homeostasis and virulence in the pathogen Salmonella enterica serovar Typhimurium. Moreover, it likely reflects that Salmonella requires dnaK, but not J-domain cochaperone-encoding genes, for survival against cytoplasmic Mg²⁺ starvation and expresses genes only when needed. Thus, the specific condition impacting protein homeostasis determines the coordinate versus differential expression of molecular chaperones and cochaperones.