## 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_

Table 2: Uniquely enriched KEGG (Kyoto Encyclopedia of Gene and Genome) pathway terms corresponding to the differentially expressed proteins. cellular growth [36]. Apart from regulating bacterial growth, AckA is also considered abroad-spectrum novel drug target in several pathogenic bacteria [37]. The cAMP modulation also controls the molecular complexes that regulate coordination between carbon metabolism and other nutrient homeostasis that are essential for the growth and survival of _E. coli_[1]. The downregulation of glutamine synthetase (GlnA; \(\sim\)2 fold) and a specific potassium ion (K\({}^{+}\))-binding protein (Kbp; \(\sim\)2.5 fold) was observed after BL illumination (Figure 2B; Table 1). In many bacteria, GlnA is an important enzyme of the central nitrogen metabolic circuit other than glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH), which are responsible for the assimilation of ammonia/ammonium (NH4\({}^{+}\)) into glutamine [38]. In bacteria, the glutamate/glutamine ratio serves as a sensor of external N availability and is essential for their proper growth and sustenance [39]. [PERSON] et al. [38] reported a _glnA_ mutant SK3112 that showed slow growth on NH4\({}^{+}\) as an N-source due to a limiting internal glutamine pool. In adverse conditions, GlnA in coordination with Cdk and Ack also provide a phosphorelay system, which behaves as a potent ATP generator [40]. This phospho-network helps in acquiring ATP by oxidative phosphorylation for bacterial growth in adverse conditions [41]. Potassium ion is involved in several key processes like the maintenance of intracellular pH and osmolarity [42]. The cytoplasmic protein Kbp, a member of RpoS regulon, is essential for normal growth in the presence of high levels of external K\({}^{+}\). [PERSON] et al. [42] compared the growth of wild-type and \(\Delta\)kbp strains at different K\({}^{+}\) concentrations and found that at higher K\({}^{+}\) concentrations, a mutation in kbp resulted in a delayed exponential phase. Non-canonical elongation factor (EF-P) was downregulated (\(\sim\)2.5 fold) in _E. coli_ illuminated with BL (Figure 2B; Table 1). In many organisms, short sequences ending with a Pro residue (PPG, PPP, or longer Pro clusters) in nascent peptides may lead to ribosome pausing (stall the translation process), which can be alleviated by EF-P [43; 44]. The regulated synthesis of proteins having terminal Pro residues in the nascent peptide by EF-P is essential for different processes. Inactivation of EF-P or its modifying enzymes YjeA or YjeK causes pleiotropic phenotypes, including a reduced growth rate and motility [45]. Growth curve analysis of LgAC-expressing _E. coli_ incubated in the dark and illuminated with BL was performed, and the results obtained showed a reduction in the growth of BL-illuminated _E. coli_ cells (Figure 3A). The untransformed _E. coli_ cells (without LgAC expression; negative control) did not show any pronounced changes in growth between the cells grown in the dark and under BL illumination (Figure S3). For _E. coli_, it is already established that glucose modulates the intracellular concentration of cAMP [46]; therefore, we considered the glucose-supplemented condition as a positive control. When the growth of _E. coli_ cells supplemented with glucose (0.1%) was compared with dark-adapted and BL-illuminated _E. coli_ cells expressing LgAC, the growth of _E. coli_ cells in the glucose-supplemented condition is comparable to dark-adapted cells (Figure S3). In _E. coli_, adenylate cyclase catalyzes the cyclization of adenosine triphosphates (ATP) into cyclic adenosine monophosphates (cAMP) by releasing a pyrophosphate (PPi) molecule. Therefore, activation of adenylate cyclase and cAMP generation may indirectly inhibit bacterial growth by reducing cellular ATP levels.