The CRISPR-Cas System differentially regulates surface-attached and pellicle-biofilm in Salmonella enterica serovar Typhimurium

CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) system has been studied for its role in biofilm regulation and expression of outer membrane proteins in Salmonella. We investigated the CRISPR-Cas mediated biofilm regulation in Salmonella enterica serovar Typhimurium by deleting CRISPR-Cas components, ΔcrisprI, ΔcrisprII, ΔΔcrisprI crisprII, and Δcas op. We determined that the system positively regulates surface-biofilm while inhibiting pellicle-biofilm. In knockout strains, flagellar (fliC, flgK) and curli (csgA) genes were repressed, causing reduced surface-biofilm. Conversely, they displayed altered pellicle-biofilm architecture possessing bacterial multilayers and a denser ECM with enhanced cellulose and lesser Curli, ergo weaker pellicle. The intracellular cellulose concentration was less in the knockout strains due to upregulation of bcsC, necessary for cellulose export. We hypothesized that exported cellulose integrates into the pellicle. We determined that crp is upregulated in the knockout strains, thereby inhibiting the expression of csgD, hence csgA and bcsA. The conflicting upregulation of bcsC, the last gene of bcsABZC operon, could be independently regulated by the CRISPR-Cas system owing to a partial match between the CRISPR-spacers and bcsC gene. The CRP-mediated regulation of the flagellar genes in the knockout strains was probably circumvented through the regulation of Yddx governing the availability of σ28 factor that further regulates class3 flagellar genes (fliC, flgK). Additionally, the variations in the LPS profile and expression of LPS-related genes (rfaC, rfbG, rfbI) in the knockout strains could also contribute to the altered pellicle architecture. Collectively, we establish that the CRISPR-Cas system differentially regulates the formation of surface-attached and pellicle-biofilm. ### Competing Interest Statement The authors have declared no competing interest.