GlcA-mediated glycerol-3-phosphate synthesis contributes to the oxidation resistance of Aspergillus fumigatus via decreasing the cellular ROS.

Fungi activate corresponding metabolic pathways in response to different carbon sources to adapt to different environments. Previous studies have shown that the glycerol kinase GlcA that phosphorylates glycerol to the intermediate glycerol-3-phosphate (G3P) is required for the growth of Aspergillus fumigatus when glycerol is used as the sole carbon source. The present study identified there were two putative glycerol kinases, GlcA and GlcB, in A. fumigatus but glycerol activated only glcA promoter but not glcB promoter, although both glcA and glcB could encode glycerol kinase. Under normal culture conditions, the absence of glcA caused no detectable colony phenotypes on glucose and other tested carbon sources except glycerol, indicating dissimilation of glucose and these tested carbon sources bypassed requirement of glcA. Notably, the oxidative stress agent H2O2 on the background of glucose medium clearly induced GlcA expression and promoted G3P synthesis. Deletion and overexpression of glcA elicited sensitivity and resistance to oxidative stress agent H2O2, respectively, accompanied by decrease and increase of G3P production. In addition, the sensitivity to oxidative stress in the glcA mutant was probably associated with dysfunction of mitochondria with a decreased mitochondrial membrane potential and an abnormal accumulation of the cellular reactive oxygen species (ROS). Furthermore, overexpressing the glycerol-3-phosphate dehydrogenase GfdA thatcatalyzes the reduction of dihydroxyacetone phosphate (DHAP) to G3P rescued phenotypes of the glcA null mutant to H2O2. Therefore, the present study suggests that GlcA-involved G3P synthesis participates in oxidative stress tolerance of A. fumigatus via regulating the cellular ROS level.

Tripartite Regulation of the glpFKD Operon Involved in Glycerol Catabolism by GylR, Crp, and SigF in Mycobacterium smegmatis.

The glpD (MSMEG_6761) gene encoding glycerol-3-phosphate dehydrogenase was shown to be crucial for M. smegmatis to utilize glycerol as the sole carbon source. The glpD gene likely forms the glpFKD operon together with glpF and glpK, encoding a glycerol facilitator and glycerol kinase, respectively. The gylR (MSMEG_6757) gene, whose product belongs to the IclR family of transcriptional regulators, was identified 182 bp upstream of glpF It was demonstrated that GylR serves as a transcriptional activator and is involved in the induction of glpFKD expression in the presence of glycerol. Three GylR-binding sites with the consensus sequence (GKTCGRC-N3-GYCGAMC) were identified in the upstream region of glpF by DNase I footprinting analysis. The presence of glycerol-3-phosphate was shown to decrease the binding affinity of GylR to the glpF upstream region with changes in the quaternary structure of GylR from tetramer to dimer. Besides GylR, cAMP receptor protein (Crp) and an alternative sigma factor, SigF, are also implicated in the regulation of glpFKD expression. Crp functions as a repressor, while SigF induces expression of glpFKD under energy-limiting conditions. In conclusion, we suggest here that the glpFKD operon is under the tripartite control of GylR, SigF, and Crp, which enables M. smegmatis to integrate the availability of glycerol, cellular energy state, and cellular levels of cAMP to exquisitely control expression of the glpFKD operon involved in glycerol metabolism.IMPORTANCE Using genetic approaches, we first revealed that glycerol is catabolized through the glycolytic pathway after conversion to dihydroxyacetone phosphate in two sequential reactions catalyzed by glycerol kinase (GlpK) and flavin adenine dinucleotide (FAD)-containing glycerol-3-phosphate dehydrogenase (GlpD) in M. smegmatis Our study also revealed that in addition to the GylR transcriptional activator that mediates the induction of the glpFKD operon by glycerol, the operon is regulated by SigF and Crp, which reflect the cellular energy state and cAMP level, respectively.

Glycerol kinase 2 is essential for proper arrangement of crescent-like mitochondria to form the mitochondrial sheath during mouse spermatogenesis.

The mitochondrial sheath is composed of mitochondria that coil tightly around the midpiece of sperm flagellum. These mitochondria are recruited from the cytoplasm to the flagellum late in spermatogenesis. Initially, recruited mitochondria are spherical-shaped but then elongate laterally to become crescent-like in shape. Subsequently, crescent-like mitochondria elongate continuously to coil tightly around the flagellum. Recently, disorganization of the mitochondrial sheath was reported in Glycerol kinase 2 (Gk2) disrupted mice. To analyze the disorganization of the mitochondrial sheath further, we generated Gk2-deficient mice using the CRISPR/Cas9 system and observed sperm mitochondria in testis using a freeze-fracture method with scanning electron microscopy. Gk2-disrupted spermatids show abnormal localization of crescent-like mitochondria, in spite of the initial proper alignment of spherical mitochondria around the flagellum, which causes abnormal mitochondrial sheath formation leading to exposure of the outer dense fibers. These results indicate that GK2 is essential for proper arrangement of crescent-like mitochondria to form the mitochondrial sheath during mouse spermatogenesis.

Aquaporin 7 expression in postimplantation mouse uteri: a potential role for glycerol transport in uterine decidualization.

The aquaglyceroporin aquaporin 7 (AQP7) is dynamically expressed in mouse uteri undergoing decidualization after implantation. The expansion of AQP7 during uterine decidualization is associated with elevated uterine glycerol accumulation and glycerol kinase expression, suggesting that glycerol might be a potential energy substrate involved in the process of decidualization.