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. 2023 Mar 22;42(12):e112514. doi: 10.15252/embj.2022112514

Figure 3. LOV histidine kinase LOV‐1477 phosphorylates diguanylate cyclase DGC‐2902 and facilitates the production of c‐di‐GMP.

Figure 3

  1. The domain annotations of proteins containing receiver (REC) domains. The asterisks highlight the proteins that interact with LOV‐1477 based on the results below.
  2. Detection of interaction between LOV‐1477 and the predicted proteins containing REC domains. Assays were done on MacConkey with 1% maltose and X‐gal‐LB indicator plate (LB broth with 40 μg ml−1 X‐gal) with 0.5 mM IPTG. Reporter strains of E. coli BTH101 were transformed, respectively, using T18 and T25 fusion constructs to exclude self‐activation. The E. coli BTH101 strain harboring pKNT25‐Zip and pCH363‐Zip plasmids was performed as the positive control.
  3. Measurement of the β‐galactosidase activity of E. coli BTH101 harboring pKNT25‐LOV1477 and pCH363 plasmids containing different REC domains of E. flavus 21‐3. The β‐galactosidase activity of the E. coli BTH101 harboring respectively used vector was used as the negative control. The β‐galactosidase activity of E. coli BTH101 harboring pKNT25‐Zip and pCH363‐Zip plasmids was measured as the positive control (N = 3 biological replicates).
  4. The relative expression of dgc‐2902 when E. flavus 21‐3 was cultivated under blue light for 36, 48, 60, and 72 h in comparison to when E. flavus 21‐3 was cultivated in the dark (N = 3 technical replicates).
  5. Comparison of the intracellular c‐di‐GMP concentration in E. coli BL21(DE3) with and without the overexpression of DGC‐2902, cultivated for 24 h. The c‐di‐GMP concentration of E. coli BL21(DE3) transformed with the empty vector was normalized as 1 (N = 3 biological replicates).
  6. Comparison of the intracellular c‐di‐GMP concentration in E. flavus 21‐3 cultivated for 36 h under blue light and in the dark. The c‐di‐GMP concentration of E. flavus 21‐3 cultivated in the dark was normalized as 1 (N = 3 biological replicates).
  7. Comparison of the intracellular c‐di‐GMP concentration in E. coli BL21(DE3) co‐expressing LOV‐1477 and DGC‐2902 after cultivation for 36 h under blue light and in the dark. The c‐di‐GMP concentration of this strain cultivated in the dark was normalized as 1 (N = 3 biological replicates).
  8. Comparison of the intracellular c‐di‐GMP concentration in E. coli BL21(DE3) co‐expressing LOV‐1477 mutant protein (with 185His mutated to Gln) and DGC‐2902, or co‐expressing LOV‐1477 and DGC‐2902 mutant protein (with 69Asp mutated to Gly), after cultivation for 36 h in the blue light and dark. The c‐di‐GMP concentration of the strains cultivated in the dark was normalized as 1 (N = 3 biological replicates).

Data information: In (C–H), data were presented as mean ± SD. Student's t‐test was performed. The P‐value for each test is presented. And n.s. means no significance.

Source data are available online for this figure.