Moonlighting activity of threonine synthase in cyanobacterial cell death

Threonine, an essential amino acid for translation, exhibits toxicity toward slow-growing cyanobacteria. This prompted us to perform transcriptomic and proteomic analyses using Microcystis aeruginosa cells treated with threonine, revealing a noteworthy upregulation of thrC, annotated as pyridoxal 5'-phosphate (PLP)-dependent threonine synthase. PyMoL-based structural prediction and in vitro biochemical assays unveiled the moonlighting functions of ThrC in both threonine biosynthesis and deamination. The production of α-ketobutyrate from threonine, both in vivo and in vitro, provided strong support for the role of ThrC as a threonine deaminase. The kinetics of ThrC as a threonine synthase using O-phospho-homoserine were consistent with the Michaelis-Menten kinetics of ThrC in Thermus thermophilus, albeit with a slightly lower V_max value. However, the in vitro deaminase activity of ThrC, exhibiting sigmoidal kinetics, suggests the potential production of α-ketobutyrate under threonine oversupply conditions in slow-growing cyanobacteria. The phylogenetic lineage of our ThrC is positioned distinctively apart from canonical threonine synthase and threonine deaminase. Gene duplication and subsequent divergence of thrC, followed by gene deletion, could have contributed to the possession of moonlighting ThrC protein in aquatic bacteria, including cyanobacterial lineages. Our metabolomic data revealed that the presence of threonine and α-ketobutyrate disrupted the balance of amino acids (alanine and methionine) and DNA biosynthetic pathways via feedback inhibition. Interestingly, the addition of exogenous alanine or methionine could alleviate threonine toxicity. Our data revealed that threonine does not uniformly support the growth of freshwater cyanobacteria to the same extent, but rather can be toxic to certain groups within this bacterial family.

The cellular stress induced by excessive amino acids in cyanobacterial lineages remains unclear. Amino acid-sensitive Microcystis aeruginosa, which lacks a complete amino acid metabolic pathway in its genome, presents a promising opportunity for investigating this phenomenon. Threonine treatment proves to be toxic to M. aeruginosa cells, causing stress on translation and energy generation due to amino acid imbalance. This imbalance is evident in transcriptome, proteome, and metabolome data. The amino acid imbalance resulting from threonine uptake impairs cell envelope integrity, leading to increased permeability and decreased transpeptidase activity in cells. Understanding the cell death mechanisms of this threonine-sensitive cyanobacterium provides insights into the molecular mechanisms underlying the death of nutrient-sensitive oligotrophic bacteria under nutrient-rich conditions.