This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
Binding of ligands to macromolecules changes their physicochemical and enzymatic characteristics. Cyclic di-GMP is a second messenger involved in motility/sessility and acute/chronic infection life style transition. Although the GGDEF domain, predominantly a diguanylate cyclase, represents one of the most abundant bacterial domain superfamilies, the number of cyclic di-GMP receptors falls short. To facilitate screening for cyclic di-nucleotide binding proteins, we describe a non-radioactive, matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF)-based modification of the widely applied differential radial capillary action of ligand assay (DRaCALA). The results of this assay suggest that the diguanylate cyclase/ phosphodiesterase variant YciR Fec101 , but not selected catalytic mutants, bind cyclic di-GMP.
Highlights• Cyclic di-nucleotides are ubiquitous second messengers in bacteria. However, few receptors have been identified.• Previous screening of cell lysates by differential radial capillary action of ligand assay (DRaCALA) using radioactive ligand identified cyclic di-nucleotide binding proteins.• A MALDI-TOF-based DRaCALA was developed to detect cyclic di-nucleotide binding as a non-radioactive alternative.• Known cyclic di-GMP binding proteins were verified and potential cyclic di-GMP binding proteins were identified.
Rdar biofilm formation of Salmonella typhimurium and Escherichia coli is a common ancient multicellular behavior relevant in cell–cell and inter-organism interactions equally, as in interaction with biotic and abiotic surfaces. With the expression of the characteristic extracellular matrix components amyloid curli fimbriae and the exopolysaccharide cellulose, the central hub for the delicate regulation of rdar morphotype expression is the orphan transcriptional regulator CsgD. Gre factors are ubiquitously interacting with RNA polymerase to selectively overcome transcriptional pausing. In this work, we found that GreA/GreB are required for expression of the csgD operon and consequently the rdar morphotype. The ability of the Gre factors to suppress transcriptional pausing and the 147 bp 5′-UTR of csgD are required for the stimulatory effect of the Gre factors on csgD expression. These novel mechanism(s) of regulation for the csgD operon might be relevant under specific stress conditions.
Expression of rdar (red, dry, and rough) colony morphology-based biofilm formation in Escherichia coli is highly variable. To investigate the molecular mechanisms of semi-constitutive rdar morphotype formation, we compared their cyclic di-GMP turnover protein content and variability to the highly regulated, temperature-dependent morphotype of the historical and modern ST10 isolates E. coli MG1655 and Fec10, respectively. Subsequently, we assessed the effects of cyclic di-GMP turnover protein variants of the EAL phosphodiesterases YcgG and YjcC and the horizontally transferred diguanylate cyclase DgcX on biofilm formation and motility. The two YcgG variants with truncations of the N-terminal CSS signaling domain were oppositely effective in targeting downregulation of rdar biofilm formation compared to the full-length reference protein. Expression of the C-terminal truncated variants YjcCFec67 and YjcCTob1 showed highly diminished apparent phosphodiesterase activity compared to the reference YjcCMG1655. For YjcCFec101, substitution of the C-terminus led to an apparently inactive enzyme. Overexpression of the diguanylate cyclase DgcX contributed to upregulation of cellulose biosynthesis but not to elevated expression of the major biofilm regulator csgD in the “classical” rdar-expressing commensal strain E. coli Fec10. Thus, the c-di-GMP regulating network is highly complex with protein variants displaying substantially different apparent enzymatic activities.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.