In recent years, our understanding of the many physiological, biochemical, and molecular functions and attributes of vitamin D-binding protein (DBP) has seen exciting and significant advances. Since its identification in 1959, many important functions of this abundant serum protein have been discovered. These range from the transport of vitamin D metabolites to possible roles in the immune system and host defense. With these discoveries, many questions regarding the biology of DBP have been raised and many remain to be answered. Our current understanding of the classic and less-recognized activities of DBP is discussed here.

The transcription factor AmrZ regulates genes important for P. aeruginosa virulence, including type IV pili, extracellular polysaccharides, and the flagellum; however, the global effect of AmrZ on gene expression remains unknown, and therefore, AmrZ may directly regulate many additional genes that are crucial for infection. Compared to the wild type strain, a ΔamrZ mutant exhibits a rugose colony phenotype, which is commonly observed in variants that accumulate the intracellular second messenger cyclic diguanylate (c-di-GMP). Cyclic di-GMP is produced by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). We hypothesized that AmrZ limits the intracellular accumulation of c-di-GMP through transcriptional repression of gene(s) encoding a DGC. In support of this, we observed elevated c-di-GMP in the ΔamrZ mutant compared to the wild type strain. Consistent with other strains that accumulate c-di-GMP, when grown as a biofilm, the ΔamrZ mutant formed larger microcolonies than the wild-type strain. This enhanced biofilm formation was abrogated by expression of a PDE. To identify potential target DGCs, a ChIP-Seq was performed and identified regions of the genome that are bound by AmrZ. RNA-Seq experiments revealed the entire AmrZ regulon, and characterized AmrZ as an activator or repressor at each binding site. We identified an AmrZ-repressed DGC-encoding gene (PA4843) from this cohort, which we named AmrZ dependent cyclase A (adcA). PAO1 overexpressing adcA accumulates 29-fold more c-di-GMP than the wild type strain, confirming the cyclase activity of AdcA. In biofilm reactors, a ΔamrZ ΔadcA double mutant formed smaller microcolonies than the single ΔamrZ mutant, indicating adcA is responsible for the hyper biofilm phenotype of the ΔamrZ mutant. This study combined the techniques of ChIP-Seq and RNA-Seq to define the comprehensive regulon of a bifunctional transcriptional regulator. Moreover, we identified a c-di-GMP mediated mechanism for AmrZ regulation of biofilm formation and chronicity. Pathogenic bacteria such as Pseudomonas aeruginosa utilize a wide variety of systems to sense and respond to the changing conditions during an infection. When a stress is sensed, signals are transmitted to impact expression of many genes that allow the bacterium to adapt to the changing conditions. AmrZ is a protein that regulates production of several virulence-associated gene products, though we predicted that its role in virulence was more expansive than previously described. Transcription factors such as AmrZ often affect the expression of a gene by binding and promoting or inhibiting expression of the target gene. Two global techniques were utilized to determine where AmrZ binds in the genome, and what effect AmrZ has once bound. This approach revealed that AmrZ represses the production of a signaling molecule called cyclic diguanylate, which is known to induce the formation of difficult to treat communities of bacteria called biofilms. This study also identified many novel targets of AmrZ to promote future studies of this regulator. Collectively, these data can be utilized to develop treatments to inhibit biofilm formation during devastating chronic infections.