Abstract
Protein phosphorylation especially on serine/threonine/tyrosine residues are frequent in many bacteria. This post-translational modification has been associated with pathogenicity and virulence in various species. However, only few data have been produced so far on generally recognized as safe bacteria used in food fermentations. A family of kinases known as Hanks-type kinases is suspected to be responsible for, at least, a part of these phosphorylations in eukaryotes as in bacteria. The objective of our work was to establish the first phosphoproteome of Streptococcus thermophilus, a lactic acid bacterium widely used in dairy fermentations in order to identified the proteins and pathways tagged by Ser/Thr/Tyr phosphorylations. In addition, we have evaluated the role in this process of the only Hanks-type kinase encoded in the S. thermophilus genome. We have constructed a mutant defective for the Hanks type kinase in S. thermophilus and established the proteomes and phosphoproteomes of the wild type and the mutant strains. To do that, we have enriched our samples in phosphopeptides with titane beads and used dimethyl tags to compare phosphopeptide abundances. Peptides and phosphopeptides were analyzed on a last generation LC-MS/MS system. We have identified and quantified 891 proteins representing half of the theoretical proteome. Among these proteins, 106 contained phosphorylated peptides. Various functional groups of proteins (amino acid, carbon and nucleotide metabolism, translation, cell cycle, stress response, …) were found phosphorylated. The phosphoproteome was only weakly reduced in the Hanks-type kinase mutant indicating that this enzyme is only one of the players in the phosphorylation process. The proteins that are modified by the Hanks-type kinase mainly belong to the divisome.
Highlights
Protein post-translational modifications are frequent and diverse in bacteria
We showed that the S. thermophilus Ser/Thr/Tyr phosphoproteome is of the same order of magnitude than the other streptococci ones as peptides belonging to 106 proteins from various metabolic pathways were found phosphorylated in one bacterial growth condition
The objectives of this work were, first, to establish the phosphoproteome of S. thermophilus, the only GRAS species in the streptococci genus, second, to identify the pathways targeted by phosphorylation and third, to evaluate the role of the only Hanks-type kinase identified in the genome of this species in this process
Summary
Protein post-translational modifications are frequent and diverse in bacteria. These modifications, not genetically encoded, affect protein conformation, activity and function. They allow quick sensing and responses to environmental changes and participate in transduction networks. It targets several amino acids, but serine, threonine and tyrosine phosphorylations are the most studied, as they are stable enough to be analyzed by mass spectrometry. Kinases, belonging to a main structural family, first described by Hanks et al (1988) and widespread in both eukaryotes and procaryotes, have emerged as potentially responsible for these phosphorylations (Cousin et al, 2013). Stancik et al (2018) recently proposed to call these kinases ‘Hanks-type kinases.’
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