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Chaperone proteins are essential in all living cells to ensure protein homeostasis. Hsp90 is a major adenosine triphosphate (ATP)-dependent chaperone highly conserved from bacteria to eukaryotes. Recent studies have shown that bacterial Hsp90 is essential in some bacteria in stress conditions and that it participates in the virulence of pathogenic bacteria. In vitro, bacterial Hsp90 directly interacts and collaborates with the Hsp70 chaperone DnaK to reactivate model substrate proteins; however, it is still unknown whether this collaboration is relevant in vivo with physiological substrates. Here, we used site-directed mutagenesis on Hsp90 to impair DnaK binding, thereby uncoupling the chaperone activities. We tested the mutants in vivo in two bacterial models in which Hsp90 has known physiological functions. We found that the Hsp90 point mutants were defective to support (1) growth under heat stress and activation of an essential Hsp90 client in the aquatic bacterium Shewanella oneidensis and (2) biosynthesis of the colibactin toxin involved in the virulence of pathogenic Escherichia coli . Our study therefore demonstrates the essentiality of the direct collaboration between Hsp90 and DnaK in vivo in bacteria to support client folding. It also suggests that this collaboration already functional in bacteria has served as an evolutionary basis for a more complex Hsp70-Hsp90 collaboration found in eukaryotes.

Local activation and long-range inhibition are mechanisms conserved in self-organizing systems leading to biological patterns. A number of them involve the production by the developing cell of an inhibitory morphogen, but how this cell becomes immune to self-inhibition is rather unknown. Under combined nitrogen starvation, the multicellular cyanobacterium Nostoc PCC 7120 develops nitrogen-fixing heterocysts with a pattern of one heterocyst every 10-12 vegetative cells. Cell differentiation is regulated by HetR which activates the synthesis of its own inhibitory morphogens, diffusion of which establishes the differentiation pattern. Here, we show that HetR interacts with HetL at the same interface as PatS, and that this interaction is necessary to suppress inhibition and to differentiate heterocysts. hetL expression is induced under nitrogen-starvation and is activated by HetR, suggesting that HetL provides immunity to the heterocyst. This protective mechanism might be conserved in other differentiating cyanobacteria as HetL homologues are spread across the phylum., Competing Interests: XX, VR, DB, SC, BD, AL No competing interests declared, (© 2020, Xu et al.)

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., Competing Interests: Competing interestsThe authors declare no competing interests.

The activity of the PrkC protein kinase is regulated in a sophisticated manner in Bacillus subtilis cells. In spores, in the presence of muropeptides, PrkC stimulates dormancy exit. The extracellular region containing PASTA domains binds peptidoglycan fragments to probably enhance the intracellular kinase activity. During exponential growth, the cell division protein GpsB interacts with the intracellular domain of PrkC to stimulate its activity. In this paper, we have reinvestigated the regulation of PrkC during exponential and stationary phases. We observed that, during exponential growth, neither its septal localization nor its activity are influenced by the addition of peptidoglycan fragments or by the deletion of one or all PASTA domains. However, Dynamic Light Scattering experiments suggest that peptidoglycan fragments bind specifically to PrkC and induce its oligomerization. In addition, during stationary phase, PrkC appeared evenly distributed in the cell wall and the deletion of one or all PASTA domains led to a non-activated kinase. We conclude that PrkC activation is not as straightforward as previously suggested and that regulation of its kinase activity via the PASTA domains and peptidoglycan fragments binding occurs when PrkC is not concentrated to the bacterial septum, but all over the cell wall in non-dividing bacillus cells.

The FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. In this work, we show that FrzCD directly binds to the nucleoid with its N-terminal positively charged tail and recruits active signaling complexes at this location. The FrzCD binding to the nucleoid occur in a DNA-sequence independent manner and leads to the formation of multiple distributed clusters that explore constrained areas. This organization might be required for cooperative interactions between clustered receptors as observed in membrane-bound chemosensory arrays.

With more than 100 antibacterial drugs at our disposal in the 1980's, the problem of bacterial infection was considered solved. Today, however, most hospital infections are insensitive to several classes of antibacterial drugs, and deadly strains of Staphylococcus aureus resistant to vancomycin--the last resort antibiotic--have recently begin to appear. Other life-threatening microbes, such as Enterococcus faecalis and Mycobacterium tuberculosis are already able to resist every available antibiotic. There is thus an urgent, and continuous need for new, preferably large-spectrum, antibacterial molecules, ideally targeting new biochemical pathways. Here we report on the progress of our structural genomics program aiming at the discovery of new antibacterial gene targets among evolutionary conserved genes of uncharacterized function. A series of bioinformatic and comparative genomics analyses were used to identify a set of 221 candidate genes common to Gram-positive and Gram-negative bacteria. These genes were split between two laboratories. They are now submitted to a systematic 3-D structure determination protocol including cloning, protein expression and purification, crystallization, X-ray diffraction, structure interpretation, and function prediction. We describe here our strategies for the 111 genes processed in our laboratory. Bioinformatics is used at most stages of the production process and out of 111 genes processed--and 17 months into the project--108 have been successfully cloned, 103 have exhibited detectable expression, 84 have led to the production of soluble protein, 46 have been purified, 12 have led to usable crystals, and 7 structures have been determined.