## 3 Results and Discussion
### LgAC is an Optogenetic Modulator of cAMP in E. coli
#### 3.2.1. _LgAC_ Regulates Growth and Energy Metabolism in _E. coli_
A comparative quantitative Western blot analysis using protein-specific antibodies of the differentially expressed protein(s) (DEP) and quantitative proteomics as well as transcriptomics (RNA-seq) would be required for targeted validation of the quantity of these protein(s). These studies would establish a direct molecular correlation (transcriptional and/or translational) between the optogenetic modulation of cAMP levels and its role in alteration of the quantity of the protein(s) associated with biofilm formation, growth, and virulence of _E. coli_. .2.2 Functional Expression of LgAC Modulates Biofilm Formation and Associated Processes in _E. coli_  
The cAMP-based regulation of the expression of genes controlling biofilm formation in many bacteria is well established [47]. In _Vibrio cholerae_ thriving under a low glucose concentration, the cAMP-CRP complex regulates biofilm formation by modulating CdgA and HapR (quorum sensing regulator) [48,49]. Biofilm formation is an important transitional event occurring in the bacterial lifestyle, wherein the inhibition of motility promotes biofilm formation. Motility-to-biofilm transition is regulated by controlling either flagellar function or genes of the flagella-biofilm regulatory network [50]. Proteomic analysis revealed a \(\sim\)2-fold downregulation of two proteins associated with the flagella-biofilm network (i.e., InfA [51] and flagellin (FliC) [52]) in BL-illuminated _E. coli_ (Figure 2B; Table 1). IhfA is one of the global regulators (GRs) of biofilm formation in _E. coli_ and modulates the expression of genes (_rpoS_, _matA_, and _csgD_) involved in the flagella-biofilm network [51]. In _E. coli_, flagella is a structural protein encoded by the _fliC_ gene, which is involved in the polymerization of the long helical filament of bacterial flagella [52]. Mutation in the _fliC_ gene causes a severe defect in biofilm formation [53]. In _Pseudomonas aeruginosa_ PAO1, the FliC protein is associated with the dispersal of the biofilm [54], where post-translational modification (phosphorylation of T27 and S28 residues) of FliC alters biofilm formation and dispersal. Proteomic analysis revealed a significant reduction (\(\sim\)2 fold) in  
Figure 3: Impact of LgAC expression on growth and biofilm formation in _E. coli_. (**A**) Growth curve of _E. coli_ expressing LgAC in the dark and BL. Growth was estimated at 600 nm. (**B**) Biofilm formation by _E. coli_ expressing LgAC in the dark and BL. The biofilm formation of _E. coli_ was estimated at 550 nm. These data are presented as mean \(\pm\) SD (\(n=6\)). Statistical comparisons were performed using ANOVA by [PERSON]’s b test using SPSS version 16.0. Asterisks (*) represents the level of significance difference as compared to control: **\(p<0.01\)** and **”ns”**, not significant. (**C**) Visualization of biofilm formation after staining with safranin. (**D**) Visualization of biofilm formation using a confocal microscopy. For each representative image, DIC and merged images are also presented. White arrowhead marked the biofilm formation. Scale bars represent 10 μm. the expression of the YeaG protein in BL-illuminated _E. coli_ (Figure 2B; Table 1). [PERSON] et al. [55] reported the _yeaG_ gene as a member of the _rpoS_ regulon that plays a crucial role in biofilm formation and its regulation in _Salmonella enterica_ Serovar Typhimurium. Further, _yeaG_ was identified as a putative stress response gene that was associated with the stress response occurring during biofilm growth [56]. A c-di-GMP receptor protein, YajQ, showed a significant decrease (~2 fold) in expression in BL-illuminated _E. coli_ (Figure 2B; Table 1). [PERSON] and coworkers performed an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads and identified the YajQ family protein as a potential cyclic di-GMP receptor, and mutation of the gene manifested a reduction in biofilm formation [57]. A reduction (~2 fold) in expression of the HfIC protein (FtsH modulator) was observed in _E. coli_ after BL illumination (Figure 2B; Table 1).