Post-translational modifications via serine/threonine phosphorylation and GpsB in Streptococcus mutans

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Post-translational modifications (PTMs), particularly protein phosphorylation, are crucial for regulating bacterial physiology.
• Streptococcus mutans relies on phosphorylation for essential cellular processes.

Purpose of the Study:

• To conduct the first comprehensive phosphoproteomic analysis of Streptococcus mutans under non-stressed conditions.
• To investigate the roles of the serine/threonine protein kinase (PknB) and phosphatase (PppL) in regulating protein phosphorylation.
• To identify substrates and signaling pathways controlled by PknB and PppL.

Main Methods:

• Tandem mass tag (TMT)-based mass spectrometry was employed for phosphoproteomic analysis.
• Phosphopeptide enrichment techniques were utilized to identify phosphorylated sites.
• Proteomic and phosphoproteomic analyses were performed on wild-type and knockout mutants (ΔpknB, ΔpppL) of S. mutans.

Main Results:

• Identified 231 high-confidence phosphosites on 131 proteins, indicating widespread O-phosphorylation.
• Phosphorylated proteins are involved in translation, carbohydrate metabolism, and cell cycle regulation.
• Analysis of ΔpknB and ΔpppL mutants revealed distinct sets of kinase and phosphatase substrates, including DivIVA, MapZ, MltG, and ribosomal proteins.
• Discovered that GpsB repression leads to lethal defects rescued by a pppL suppressor mutation, restoring DivIVA phosphorylation and highlighting the PknB/PppL signaling axis.

Conclusions:

• O-phosphorylation plays a broad role in regulating core cellular functions in Streptococcus mutans.
• PknB and PppL are key regulators of bacterial phosphosignaling, with overlapping yet distinct substrate specificities.
• The PknB/PppL signaling axis, potentially modulated by GpsB, is essential for maintaining phosphorylation of critical targets in S. mutans.