Pseudomonas aeruginosa infections have reached a “critical” threat status making novel therapeutic approaches required.

The Joint Safety Team (JST) was conceived in 2012 by the Departments of Chemical Engineering and Materials Science and Chemistry at the University of Minnesota and the Dow Chemical Company as a model student-led safety organization. The JST initiative was aimed at improving academic safety through four core areas: compliance, awareness, resources, and education. Since its inception, the JST has taken great strides to develop a culture of peer-led safety at Minnesota. We describe the evolution of the structure of the organization over the last 8 years and the innovative methodologies employed by the JST to educate and evaluate safety in academic laboratories. The continuous efforts of the student members of the JST have enabled the organization to be recognized as a leader in peer-to-peer safety. The Minnesota model of “inform and reform” is now being adopted at other academic institutions to develop safety organizations emulating the JST.

While two-component systems (TCSs), composed of a sensor histidine kinase (HK) and a response regulator, are the main signaling pathways in bacteria, global TCS activity remains poorly described. Here, we report the kinetic parameters of the HK autophosphorylation reaction using previously uncharacterized γ-phosphate-modified ATP analogues to further elucidate their utility as activity-based probes for global TCS analysis. Given the increased stability of thiophosphorylated histidine in comparison to that of the native phosphoryl modification, which is attributed to the decreased electrophilicity of this moiety, we anticipated that ATPγS may be turned over much more slowly by the HKs. Surprisingly, we found this not to be the case, with the turnover numbers decreasing <1 order of magnitude. Instead, we found that alkylation of the thiophosphate had a much more dramatic effect on turnover and, in one case, the binding affinity of this substrate analogue (BODIPY-FL-ATPγS).

Modified nucleoside triphosphates (NTPs) are invaluable tools to probe bacterial enzymatic mechanisms, develop novel genetic material, and engineer drugs and proteins with new functionalities.

Phosphorylation is an essential protein modification and is most commonly associated with hydroxyl-containing amino acids via an adenosine triphosphate (ATP) substrate. The last decades have brought greater appreciation to the roles that phosphorylation of myriad amino acids plays in biological signaling, metabolism, and gene transcription. Histidine phosphorylation occurs in both eukaryotes and prokaryotes but has been shown to dominate signaling networks in the latter due to its role in microbial two-component systems. Methods to investigate histidine phosphorylation have lagged behind those to study serine, threonine, and tyrosine modifications due to its inherent instability and the historical view that this protein modification was rare. An important strategy to overcome the reactivity of phosphohistidine is the development of substrate-based probes with altered chemical properties that improve modification longevity but that do not suffer from poor recognition or transfer by the protein. Here, we present combined experimental and computational studies to better understand the molecular requirements for efficient histidine phosphorylation by comparison of the native kinase substrate, ATP, and alkylated ATP derivatives. While recognition of the substrates by the histidine kinases is an important parameter for the formation of phosphohistidine derivatives, reaction sterics also affect the outcome. In addition, we found that stability of the resulting phosphohistidine moieties correlates with the stability of their hydrolysis products, specifically with their free energy in solution. Interestingly, alkylation dramatically affects the stability of the phosphohistidine derivatives at very acidic pH values. These results provide critical mechanistic insights into histidine phosphorylation and will facilitate the design of future probes to study enzymatic histidine phosphorylation.