## 4 Conclusions  
The establishment of a new optogenetic modulation is an innovative approach to reprogram gene expression and cell signaling with spatiotemporal precision. A variety of optogenetic tools have been developed for controlling neural activity, behavioral response, and cellular events in cells and the whole organism. In bacteria, cAMP signaling regulates a variety of crucial processes essential for its growth, survival, and pathogenesis. We established optogenetic modulation of cAMP in a model bacterium (_E. coli_) and studied the optogenetically modulated cAMP-dependent bacterial processes like growth, biofilm formation, and virulence using a light-gated adenylate cyclase (LgAC). The blue light-dependent activation of LgAC and the corresponding increase in cellular cAMP level demonstrated a significant decrease in the growth and levels of relevant proteins (PTS, Adk, AckA, GlnA, Kbp, and EF-P). Proteins regulating the motility to biofilm transition and flagella-biofilm network were modulated upon optogenetic control of cAMP. Photoactivation of LgAC modulated the amount of IhfA, YajQ, and HfIC, which regulates crucial steps in biofilm formation. Photoinduced elevation in the cAMP level influenced the levels of proteins (ClpP, YebC, ButE, KatE, and Zur), which are responsible for conferring virulence. ClpP and YebC proteins are known to regulate different aspects of bacterial virulence and pathogenesis. ButE and Zur represent the group of proteins that helps pathogens counter oxidative stress imposed by the host immune system. It is evident that LgAC is an optogenetic photoswitch, which can modulate cAMP-dependent bacterial growth, biofilm formation, and pathogenesis simply by illumination with blue light. The established optogenetic modulation of bacterial cAMP opens avenues for studying cAMP-driven physiological aspects, including the host-pathogen signaling of bacterial systems. The systematic utilization of the established method and more extensively designed  
Figure 4: Growth-biofilm-pathogenicity protein network. Protein–protein interaction analysis was performed by using String version 11 ([[https://string-db.org/](https://string-db.org/)]([https://string-db.org/](https://string-db.org/));) and further analyzed by Cytoscape 3.4.1 applying the betweenness centrality algorithm. Color range represents betweenness values in the network. Protein network framed under blue, pink, and green lines belongs to biofilm formation, virulence, and growth, respectively. Blue, pink, and green solid arrows represent proteins that showed differential expression in quantitative proteomic analysis of LgAC-expressing _E. coli_ incubated in the dark and under blue light illumination, respectively. experiments regarding the bacterial growth, biofilm, survival, and virulence might help in providing a road map for the identification of new targets for developing antibacterial drugs. The following are available online at [[http://www.mdpi.com/2076-3417/10/16/5535/s1](http://www.mdpi.com/2076-3417/10/16/5535/s1)]([http://www.mdpi.com/2076-3417/10/16/5535/s1](http://www.mdpi.com/2076-3417/10/16/5535/s1)), Figure S1: Multiple sequence alignment of the BLUF (blue light using flavin) domain (**A**), linker region (**B**), and cyclase domain (**C**) from different organisms with that of the well-characterized BLUF-Cyclase proteins from _Oscilatoria accuminata_ depicting the functionally important amino acids in the BLUF domain (red star represent the amino acids important for photocycle and photodynamics of the BLUF while the orange square denotes the residues essential for the flavin binding pocket), in the cyclase domain [green stars represent important amino acids required for ATP binding, red arrows depict metal-binding residues (MBR), blue arrows correspond to the essential adenine-binding residues (ABR), and orange arrows denote transition state-stabilizing residues (TSSR)], respectively.