An Ultrasensitive Biosensor for Probing Subcellular Distribution and Mitochondrial Transport of l-2-Hydroxyglutarate.

l-2-Hydroxyglutarate (l-2-HG) is a functionally compartmentalized metabolite involved in various physiological processes. However, its subcellular distribution and mitochondrial transport remain unclear owing to technical limitations. In the present study, an ultrasensitive l-2-HG biosensor, sfLHGFRH, composed of circularly permuted yellow fluorescent protein and l-2-HG-specific transcriptional regulator, is developed. The ability of sfLHGFRH to be used for analyzing l-2-HG metabolism is first determined in human embryonic kidney cells (HEK293FT) and macrophages. Then, the subcellular distribution of l-2-HG in HEK293FT cells and the lower abundance of mitochondrial l-2-HG are identified by the sfLHGFRH-supported spatiotemporal l-2-HG monitoring. Finally, the role of the l-glutamate transporter SLC1A1 in mitochondrial l-2-HG uptake is elucidated using sfLHGFRH. Based on the design of sfLHGFRH, another highly sensitive biosensor with a low limit of detection, sfLHGFRL, is developed for the point-of-care diagnosis of l-2-HG-related diseases. The accumulation of l-2-HG in the urine of patients with kidney cancer is determined using the sfLHGFRL biosensor.

Sulfane Sulfur is an intrinsic signal activating MexR-regulated antibiotic resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO1, an opportunistic human pathogen, deploys several strategies to resist antibiotics. It uses multidrug efflux pumps, including the MexAB-OprM pump, for antibiotic resistance, and it also produces hydrogen sulfide (H2 S) that provides some defense against antibiotics. MexR functions as a transcriptional repressor of the mexAB-oprM operon. MexR responds to oxidative stresses caused by antibiotic exposure, and it also displays a growth phase-dependent derepression of the mexAB-oprM operon. However, the intrinsic inducer has not been identified. Here, we report that P. aeruginosa PAO1 produced sulfane sulfur, including glutathione persulfide and inorganic polysulfide, produced from either H2 S oxidation or from L-cysteine metabolism. Sulfane sulfur directly reacted with MexR, forming di- and trisulfide cross-links between two Cys residues, to derepress the mexAB-oprM operon. Levels of cellular sulfane sulfur and mexAB-oprM expression varied during growth, and both reached the maximum during the stationary phase of growth. Thus, self-produced H2 S and sulfane sulfur may facilitate antibiotic resistance via inducing the expression of antibiotic resistance genes.

FisR activates σ54 -dependent transcription of sulfide-oxidizing genes in Cupriavidus pinatubonensis JMP134.

Some heterotrophic bacteria are able to oxidize sulfide (H2 S, HS- and S2- ) to sulfite and thiosulfate via polysulfide. The genes coding for the oxidation enzymes in Cupriavidus pinatubonensis JMP134 have recently been identified; however, their regulation is unknown. A regulator gene is adjacent to the operon of the sulfide-oxidizing genes, encoding a σ54 -dependent transcription factor (FisR) with three domains: an R domain, an AAA+ domain and a DNA-binding domain. Here it is reported that the regulator responds to the presence of sulfide and activates the sulfide-oxidizing genes. FisR binds to its cognate operator at -114 to -135 bp of the transcription start of the operon. When polysulfide reacts with the R domain of FisR through the three conserved cysteine residues (C53, C64 and C71), FisR activates the expression of the operon. FisR is highly sensitive to polysulfide, activating σ54 -dependent transcription of sulfide-oxidizing genes for sulfide removal. Further, sequence analysis indicates that FisR-type regulators are relatively common for controlling sulfide-oxidizing genes under sulfide stress in the Proteobacteria.