Visualization of Imbalances in Sulfur Assimilation and Synthesis of Sulfur-Containing Amino Acids at the Single-Cell Level

We describe genetically encoded sensors which transmit elevated cytosolic concentrations of O-acetyl serine (OAS) and O-acetyl homoserine (OAH)—intermediates ofl -cysteine andl -methionine synthesis—into an optical output. The sensor pSenOAS3 elicits 7.5-fold-increased fluorescence in cultures of aCorynebacterium glutamicum strain that excretel -cysteine. Determination of the cytosolic OAS concentration revealed an increase to 0.13 mM, whereas the concentration in the reference strain was below the detection limit, indicating that incorporation of assimilatory sulfur is limited in the strain studied. In another strain, overexpression ofmetX encoding homoserine acetyltransferase resulted in an 8-fold increase in culture fluorescence at a cytosolic OAH concentration of 0.76 mM. We also assayed for consequences of extracellular sulfur supply and observed a graded fluorescence increase at decreasing sulfur concentrations below 400 μM. Overall, this demonstrates the usefulness of the sensors for monitoring intracellular sulfur availability. The sensors also enable monitoring at the single-cell level, and since related and close homologs of the transcription factor used in the constructed sensors are widespread among bacteria, this technology offers a new possibility of assayingin vivo for sulfur limitation and of doing this at the single-cell level.