Bacterial adaptation to cold: Conservation of a short J-domain co-chaperone and its protein partners in environmental proteobacteria.

Bacterial genomes are a huge reservoir of genes encoding J-domain protein co-chaperones that recruit the molecular chaperone DnaK to assist protein substrates involved in survival, adaptation, or fitness. The atc operon of the aquatic mesophilic bacterium Shewanella oneidensis encodes the proteins AtcJ, AtcA, AtcB, and AtcC, and all of them, except AtcA, are required for growth at low temperatures. AtcJ is a short J-domain protein that interacts with DnaK, but also with AtcC through its 21 amino acid C-terminal domain. This interaction network is critical for cold growth. Here, we show that AtcJ represents a subfamily of short J-domain proteins that (i) are found in several environmental, mostly aquatic, ß- or É£-proteobacteria and (ii) contain a conserved PX7 W motif in their C-terminal extension. Using a combination of NMR, biochemical and genetic approaches, we show that the hydrophobic nature of the tryptophan of the S. oneidensis AtcJ PX7 W motif determines the strong AtcJ-AtcC interaction essential for cold growth. The AtcJ homologues are encoded by operons containing at least the S. oneidensis atcA, atcB, and atcC homologues. These findings suggest a conserved network of DnaK and Atc proteins necessary for low-temperature growth and, given the variation in the atc operons, possibly for other biological functions.

Modulation of the RNA polymerase activity by AtcB, a protein associated with a DnaK chaperone network in Shewanella oneidensis.

Bacteria possess several molecular pathways to adapt to changing environments and to stress conditions. One of these pathways involves a complex network of chaperone proteins that together control proteostasis. In the aquatic bacterium Shewanella oneidensis, we have recently identified a previously unknown co-chaperone of the DnaK/Hsp70 chaperone system, AtcJ, that is essential for adaptation to low temperatures. AtcJ is encoded in the atcJABC operon, whose products, together with DnaK, form a protein network allowing growth at low temperature. However, how these proteins allow cold adaptation is unknown. Here, we found that AtcB directly interacts with the RNA polymerase and decreases its activity. In addition, AtcB overproduction prevents bacterial growth due to RNA polymerase inhibition. Together, these results suggest that the Atc proteins could direct the DnaK chaperone to the RNA polymerase to sustain life at low temperatures.

Cold adaptation in the environmental bacterium Shewanella oneidensis is controlled by a J-domain co-chaperone protein network.

DnaK (Hsp70) is a major ATP-dependent chaperone that functions with two co-chaperones, a J-domain protein (JDP) and a nucleotide exchange factor to maintain proteostasis in most organisms. Here, we show that the environmental bacterium Shewanella oneidensis possesses a previously uncharacterized short JDP, AtcJ, dedicated to cold adaptation and composed of a functional J-domain and a C-terminal extension of 21 amino acids. We showed that atcJ is the first gene of an operon encoding also AtcA, AtcB and AtcC, three proteins of unknown functions. Interestingly, we found that the absence of AtcJ, AtcB or AtcC leads to a dramatically reduced growth at low temperature. In addition, we demonstrated that AtcJ interacts via its C-terminal extension with AtcC, and that AtcC binds to AtcB. Therefore, we identified a previously uncharacterized protein network that involves the DnaK system with a dedicated JDP to allow bacteria to survive to cold environment.

Interplay between the Hsp90 Chaperone and the HslVU Protease To Regulate the Level of an Essential Protein in Shewanella oneidensis.

Protein synthesis, folding, and degradation are an accurately regulated process occurring in every organism and called proteostasis. This process is essential to maintain a healthy proteome since proteostasis dysregulation is responsible for devastating cellular issues. Proteostasis is controlled by a complex network of molecular chaperones and proteases. Among them, eukaryotic Hsp90, assisted by many cochaperones and the Hsp70 chaperone system, plays a major role in activating hundreds of client proteins, and Hsp90 inhibition usually leads to proteasomal degradation of these clients. In bacteria, however, the precise function of Hsp90 remains quite unclear, and only a few clients are known. Recently, we have shown that Hsp90 is essential at elevated temperature in the aquatic model bacterium Shewanella oneidensis, and we have identified a client of Hsp90, TilS, involved in tRNA modification. Here we found that two members of the proteostasis network with antagonist activities, the Hsp90 chaperone and the HslVU protease, which is considered the proteasome ancestor, together regulate the level of TilS. In particular, we show that deletion of the genes coding for the HslVU protease suppresses the growth defect of an S. oneidensis strain with hsp90 deleted, by increasing the cellular level of the essential TilS protein. These results open up new avenues for understanding how proteostasis is controlled in bacteria, and new Hsp90 clients are much needed now to confirm the interplay between Hsp90 and proteases.IMPORTANCE Maintaining a healthy proteome is essential in every living cell from bacteria to humans. For example, proteostasis (protein homeostasis) imbalance in humans leads to devastating diseases, including neurodegenerative diseases and cancers. Therefore, proteins need to be assisted from their synthesis to their native folding and ultimately to their degradation. To ensure efficient protein turnover, cells possess an intricate network of molecular chaperones and proteases for protein folding and degradation. However, these networks need to be better defined and understood. Here, using the aquatic bacterium Shewanella oneidensis as a model organism, we demonstrate interplay between two proteins with antagonist activities, the Hsp90 chaperone and the HslVU protease, to finely regulate the level of an essential client of Hsp90. Therefore, this work provides a new bacterial model to better study protein regulation and turnover, and it sheds light on how proteostasis by Hsp90 and proteases could be controlled in bacteria.