The kinases HipA and HipA7 phosphorylate different substrate pools in

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Bacterial serine-threonine protein kinase HipA regulates multidrug tolerance and persistence.
• Persistence is a dormant state induced by HipA through GltX phosphorylation, halting translation and growth.
• The HipA variant, HipA7, enhances persistence but is less effective at inhibiting cell growth.

Purpose of the Study:

• To investigate the substrate specificity of HipA and HipA7 variants.
• To elucidate the molecular mechanisms underlying the differential effects of HipA and HipA7 on bacterial persistence and growth inhibition.
• To identify endogenous substrates of HipA and HipA7 in Escherichia coli.

Main Methods:

• Overproduction of HipA and HipA7 in Escherichia coli.
• SILAC-based quantitative phosphoproteomics to identify endogenous kinase substrates.
• Kinase activity assays and autophosphorylation analysis.
• Analysis of substrate phosphorylation under different expression conditions (plasmid vs. chromosomal).

Main Results:

• GltX was confirmed as the primary substrate for both HipA and HipA7, crucial for persistence.
• HipA7 exhibited reduced kinase activity and primarily targeted GltX, especially at moderate expression levels.
• HipA phosphorylated additional substrates, including RplK and SeqA, involved in translation, transcription, and replication.
• Autophosphorylation was significantly reduced in HipA7 compared to HipA.
• Endogenously expressed HipA was inhibited by HipB, while HipA7 phosphorylated GltX and PspA.

Conclusions:

• Differences in substrate specificity, particularly HipA targeting additional proteins beyond GltX, likely explain the distinct phenotypes of HipA and HipA7.
• HipA7's enhanced persistence is primarily mediated by GltX phosphorylation, with its reduced kinase activity contributing to its specific effects.
• The study provides insights into the regulation of bacterial persistence and identifies potential targets for understanding HipA-mediated processes.