Sulfane sulfur is common inside cells, playing both regulatory and antioxidant roles. However, there are unresolved issues about its chemistry and biochemistry. We report the discovery that reactive sulfane sulfur such as polysulfides and persulfides could be detected by using resonance synchronous spectroscopy (RS 2 ). With RS 2 , we showed that inorganic polysulfides at low concentrations were unstable with a half-life about 1 min under physiological conditions due to reacting with glutathione. The protonated form of glutathione persulfide (GSSH) was electrophilic and had RS 2 signal. GSS − was nucleophilic, prone to oxidation, but had no RS 2 signal. Using this phenomenon, p K a of GSSH was determined as 6.9. GSSH/GSS − was 50-fold more reactive than H 2 S/HS − towards H 2 O 2 at pH 7.4, supporting reactive sulfane sulfur species like GSSH/GSS − may act as antioxidants inside cells. Further, protein persulfides were shown to be in two forms: at pH 7.4 the deprotonated form (R-SS - ) without RS 2 signal was not reactive toward sulfite, and the protonated form (R-SSH) in the active site of a rhodanese had RS 2 signal and readily reacted with sulfite to produce thiosulfate. These data suggest that RS 2 of sulfane sulfur is likely associated with its electrophilicity. Sulfane sulfur showed species-specific RS 2 spectra and intensities at physiological pH, which may reveal the relative abundance of a reactive sulfane sulfur species inside cells.

Three double-Cys mutant pairs--Ala273-->Cys/Met299-->Cys, Thr266-->Cys/Ile303-->Cys, and Thr266-->Cys/Ser306-->Cys--were constructed in a functional lac permease construct devoid of Cys residues, and the excimer fluorescence or electron paramagnetic resonance (EPR) was studied with pyrene- or spin-labeled derivatives, respectively. After reconstitution into proteoliposomes, excimer fluorescence is observed with mutant Ala273-->Cys/Met299-->Cys, but not with the single-Cys mutants nor with mutants Thr266-->Cys/Ile303-->Cys or Thr266-->Cys/Ser306-->Cys. Furthermore, spin-spin interaction is also observed with mutant Ala273-->Cys/Met299-->Cys, but only after the permease is reconstituted into proteoliposomes. The results provide independent support for the conclusions that helix VIII is close to helix IX and that the transmembrane helices of the permease are more loosely packed in a detergent micelle as opposed to a phospholipid bilayer.

Microorganisms often use specific autoinducers other than common metabolites for quorum sensing (QS). Herein, we demonstrated that Escherichia coli produced sulfide (H2S, HS–, and S2–) with the concentrations proportionally correlated to its cell density. We then designed synthetic gene circuits that used H2S as an autoinducer for quorum sensing. A sulfide/quinone oxidoreductase converted diffusible H2S to indiffusible hydrogen polysulfide (HS n H, n ≥ 2), and a gene regulator CstR sensed the latter to turn on the gene expression. We constructed three element libraries, with which 24 different circuits could be assembled for adjustable sensitivity to cell density. The H2S-mediated gene circuits endowed E. coli cells within the same batch or microcolony with highly synchronous behaviors. Using them we successfully constructed cell factories capable of an autonomous switch from growth phase to production phase. Thus, these circuits provide a new tool-kit for metabolic engineering and synthetic biology.