Adenylate Cyclase

The DNA fragment of a red fluorescent protein variant, mApple, utilized in a red Ca²⁺ indicator R-GECO^{12 (link)} was created by DNA synthesis from Integrated DNA Technologies (Coralville, IA, USA). It was engineered to contain SacII and EcoRI restriction enzyme sites between A150 and V151 and cloned into the pRSET-A vector. The cAMP binding domain of mEpac1 (NM_001171281, 199–358 amino acids) derived from Flamindo2 and was inserted into the SacII/EcoRI sites of mApple. To improve the performance of the indicator, a number of variants were created with the addition or deletion of linker amino acids in the both N-terminus and C-terminus of the cAMP binding domain by PCR. Random, site-directed mutations were also introduced into the construct by PCR with sense and anti-sense primers including NNK and MNN, respectively. The mutant construct that elicited the highest dynamic range was selected and named Pink Flamindo. Pink Flamindo was cloned into the pcDNA3.1 (-) vector for expression in mammalian cells. The humanised photoactivated adenylyl cyclase from Beggiatoa sp. (h_bPAC) in a pGEM-HE vector was obtained from Addgene (#28134, Cambridge, MA, USA), and was subcloned into the BglII/EcoRI sites of the pEGFP-C1 vector. The G-GECO plasmid was also obtained from Addgene (#32447). The Flamindo2 plasmid was developed in our previous study^{9 (link)}.

The BACTH system was used to assess the interactions between BlsA and BipA (53 (link)). As BlsA interacts with Fur in A. baumannii, Fur was used as a positive control for the BACTH system. Moreover, fur (473 bp) and bipA (363 bp) were cloned into pKNT25 and pKT25 vectors to fuse each protein into the adenylate cyclase subunit T25 of Bordetella pertussis. The plasmids and primers used in the BACTH system are listed in Tables S6 and S7, respectively. Each target gene (fur and bipA) was individually subcloned into the vector pKNT25 using the enzymes BamHI and HindIII to create fur-T25 and bipA-T25 fusion constructs, respectively, which had the adenylate cyclase subunit T25 at the C-terminal regions of the fused proteins. To create the T25-bipA fusion construct, bipA was subcloned into the vector pKT25, using the enzymes BamHI and EcoRI. The other subunit of adenylate cyclase, T18, was fused to BlsA in the pUT18C vector using the enzymes EcoRI and BamHI to produce the T18-blsA fusion protein. The cyaA-deleted strain E. coli BTH101 was cotransformed with the constructed plasmids pUT18C::blsA and pKNT25::bipA/fur as well as pUT18C::blsA and pKT25::bipA. Successful insertions were confirmed via the polymerase chain reaction (PCR) amplification and sequencing of the corresponding parts of the clones. The transformants were selected on LB agar plates supplemented with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (IPTG, 40 μg/mL), isopropyl-β-d-thiogalactopyranoside (X-Gal, 0.5 mM), ampicillin (100 μg/mL), and kanamycin (50 μg/mL). Colonies that were blue in color after being grown on the agar plate for 24 h were considered to have positive interactions. The β-galactosidase activity in LB broth was then assessed to quantify the interactions. Fivefold higher galactosidase activity (expressed in Miller units) than the negative control was considered to be a positive interaction (34 (link), 35 (link), 54 (link)). Statistical significance was determined using GraphPad Prism 9 (Student’s t test). Three biological replicates were used to validate the data. The cotransformation of pKNT25::fur and pUT18C::blsA into E. coli BTH101 was performed in the positive control. All of the tests were performed at least thrice at 23°C under blue light (4 W/m²) or dark conditions for 24 h.