## 3 Results and Discussion  
### LgAC is an Optogenetic Modulator of cAMP in E. coli  
The homology analysis of the BLUF domain (Figure S1A), linker region (Figure S1B), and cyclase domain (Figure S1C) from 22 BLUF cyclases was performed against the well-characterized PAC from _Oscillatoria acuninata_. The residues important for BLUF photodynamics and ATP binding pocket of the cyclase domain were mostly found to be conserved. LgAC from _Beggiatoa_ sp. showed the maximum conservation for the ATP binding pocket residues (ASP6, LEU8, ALA9, PHE10, SER11, THR12, ILE48, GLY49, ASP50, CYS51, ARG133) (Figure S1C and S2; [31]). Therefore, the LgAC from _Beggiatoa_ sp. was selected as the PAC to be used as an optogenetic tool for the regulation of cAMP-dependent bacterial physiological processes. Further, to study the impact of light-gated adenylate cyclase on cAMP-dependent processes in bacteria, the heterologous expression of recombinant LgAC was induced in _E. coli_. The LgAC was expressed in _E. coli_ cells as an N-terminal Histidine (His) tag fusion protein (5X His:LgAC). The spectroscopic analysis of the purified recombinant LgAC suggested the functional expression of LgAC in _E. coli_ (Figure 1A). In addition, Western blotting with an anti-His antibody (Figure 1B) and confocal analysis (Figure 1C-E) displayed a robust expression of recombinant LgAC in _E. coli_ cells. An ELISA-based immunoassay was performed to estimate the cAMP level in the total lysates prepared from the LgAC-expressing cells incubated under dark and BL to confirm the photoactivation of LgAC. A significant increase (\(\sim\)10 fold; \(p<0.001\)) in the cAMP level was detected in _E. coli_ after BL illumination when compared to dark-adapted cells (Figure 1F); however, insignificant cyclase activity of LgAC under dark corresponding to the basal cyclase activity in _E. coli_ cells was also observed. Label-Free Quantitative Proteomics Revealed cAMP-Dependent Modulation of Growth, Biofilm Formation, and Virulence in E. coli  
The comparative proteomics data of LgAC-carrying _E. coli_ cells incubated in the dark and illuminated with BL revealed quantitative changes in the protein profile. A total of 732 proteins were identified (Figure 2A), out of which 57 were differentially expressed, and are represented by a heat map (Figure 2B; Table 1). The KEGG pathway analysis categorically linked differentially expressed proteins (DEPs) with 29 different pathways (Figure 2C; Table 2). The DEPs involved in growth, biofilm formation, and virulence were studied in detail. Figure 1: Photoactivation of light-gated adenylate cyclase (LgAC). (**A**) Spectral properties of recombinant LgAC in the dark (solid line) and blue light-adapted (dotted line) states. A difference spectrum between blue light and dark-adapted LgAC is represented by the dashed line. (**B**) Immunoblot analysis of recombinant LgAC protein. E1, E2, and E3 are the eluted fractions of the purified recombinant LgAC probed with anti-His antibody (**C**) Visualization of intracellular expression of LgAC in _E. coli_ cells. (**D**) A differential interference contrast (DIC) image and merged images (**E**) are also presented. Scale bars represent 10 μm. (**F**) Variation in the total cAMP level in the cell lysates extracted from _E. coli_ cells expressing LgAC in the dark and BL. The data are represented as mean \(\pm\) SD and were statistically tested employing student’s \(t\) test using SPSS version 16.0. Asterisks (*) represents the level of significance of the difference as compared to the control (dark): *** \(p\) < 0.001. Figure 2: Comparative proteomics of LgAC-expressing _E. coli_ incubated in the dark and illuminated with BL. (**A**) Venn diagram representing the distribution of proteins identified by label-free comparative quantitative proteomics as total protein, dark condition specific, BL specific, and differentially expressed proteins (DEPs) using Venny 2.1. (**B**) Heat map showing comparative expression patterns of the DEPs.