Escherichia coli dh5α

S. aureus NCTC 8325, a type strain of S. aureus, was obtained from the China Medical Culture Collection Center (CMCC) and stored at −80°C. Escherichia coli DH5α was purchased from Solarbio, China (C1100) and used for plasmid construction.
NCTC 8325 colony was incubated overnight at 37°C with TSB broth in a shaker rotational speed of 200 r/min. All experiments were set up with experimental groups and blank groups. The experimental groups were stimulated with 50 µg/mL AGEs in TSB broth and cultured for 24 h. AGEs were purchased from Abcam, USA (ab51995). The blank groups were stimulated with the same concentration of bovine serum albumin (BSA) (EZ7890B203, BioFroxx, Germany).
The SigB promoter sequence was transferred into pGL4.10 vector (YouBio, China, VT1558) by restriction endonuclease sites XhoI and HindIII to express SigB promoter. The glmS gene CDS region was transferred into pcDNA3.1 and pET-28a vectors (YouBio, China, VT1001, VT1207) by restriction endonuclease sites XhoI and BamH I to express GlmS protein. The recombination products were transformed into competent E. coli DH5α cells and BL21(DE3) cells. Recombinant positive plasmids of pGL4.10 and pcDNA3.1 vectors were screened through ampicillin containing medium, while recombinant positive plasmids of pET-28a vector were screened through kanamycin containing medium.

All mutants were generated from the wild-type M. acridum strain CQMa102 (WT) and grown on 1/4 SDAY media (10‰ glucose, 5‰ yeast extract, 2.5‰ peptone and 18‰ agar, wt/vol) or SYA media (5‰ yeast extract, 0.5‰ KCl, 1‰ KH₂PO₄, 30‰ sucrose, 0.5‰ MgSO₄, 3‰ NaNO₃, 0.01‰ MnSO₄, 0.01‰ FeSO₄ and 18‰ agar, wt/vol) at 28°C. The Y2HGold and Y187 yeast strains (Clontech, Palo Alto, CA, USA) were used in the autoactivation and yeast one-hybrid assays, respectively. Escherichia coli DH5α (Solarbio, Beijing, China) and Agrobacterium tumefaciens AGL1 (Solarbio, Beijing, China) were used for the recombinant plasmid manipulations and fungal transformations, respectively.