Anionic (i.e., acidic) phospholipids such as phosphotidylglycerol (PG) and cardiolipin (CL), participate in several cellular functions. Here we review intriguing in vitro and in vivo evidence that suggest emergent roles for acidic phospholipids in regulating DnaA protein-mediated initiation of Escherichia coli chromosomal replication. In vitro acidic phospholipids in a fluid bilayer promote the conversion of inactive ADP-DnaA to replicatively proficient ATP-DnaA, yet both PG and CL also can inhibit the DNA-binding activity of DnaA protein. We discuss how cellular acidic phospholipids may positively and negatively influence the initiation activity of DnaA protein to help assure chromosomal replication occurs once, but only once, per cell-cycle. Fluorescence microscopy has revealed that PG and CL exist in domains located at the cell poles and mid-cell, and several studies link membrane curvature with sub-cellular localization of various integral and peripheral membrane proteins. E. coli DnaA itself is found at the cell membrane and forms helical structures along the longitudinal axis of the cell. We propose that there is cross-talk between acidic phospholipids in the bacterial membrane and DnaA protein as a means to help control the spatial and temporal regulation of chromosomal replication in bacteria.

DnaA oligomerizes when bound to origins of chromosomal replication. Structural analysis of a truncated form of DnaA from Aquifex aeolicus has provided insight into crucial conformational differences within the AAA+ domain that are specific to the ATP- versus ADP- bound form of DnaA. In this study molecular docking of ATP and ADP onto Escherichia coli DnaA, modeled on the crystal structure of Aquifex aeolicus DnaA, reveals changes in the orientation of amino acid residues within or near the vicinity of the nucleotide-binding pocket. Upon limited proteolysis with trypsin or chymotrypsin ADP-DnaA, but not ATP-DnaA generated relatively stable proteolytic fragments of various sizes. Examined sites of limited protease susceptibility that differ between ATP-DnaA and ADP-DnaA largely reside in the amino terminal half of DnaA. The concentration of adenine nucleotide needed to induce conformational changes, as detected by these protease susceptibilities of DnaA, coincides with the conversion of an inactive bacterial origin recognition complex (bORC) to a replication efficient pre-replication complex (pre-RC) at the E. coli chromosomal origin of replication (oriC).

Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution. Whereas ADP-DnaA was predominantly monomeric, AMP–PNP–DnaA (a non-hydrolysable ATP-analog bound-DnaA) was oligomeric, primarily dimeric. Functional studies using DnaA mutants revealed that DnaA(H136Q) is defective in initiating replication in vivo. The mutant retains high-affinity ATP binding, but was defective in producing replication-competent initiation complexes. Docking of ATP on a structure of E. coli DnaA, modeled upon the crystallographic structure of Aquifex aeolicus DnaA, predicts a hydrogen bond between ATP and imidazole ring of His136, which is disrupted when Gln is present at position 136. SAXS performed on AMP–PNP–DnaA (H136Q) indicates that the protein has lost its ability to form oligomers. These results show the importance of high ATP in DnaA oligomerization and its dependence on the His136 residue.

Fungal communities in the gut influence host immunity, yet most studies have focused on cell wall components rather than genetic materials. Here, we explore how fungal genomic DNA (gDNA) from Candida albicans, Saccharomyces cerevisiae, and Cryptococcus neoformans modulate immune responses in human CD4⁺ T cells, murine splenocytes, and THP-1-derived macrophages. We find that C. albicans gDNA promotes the development of regulatory T cells and increases IL-10, fostering immune tolerance and preserving CD4⁺ T cell viability in an inflammatory setting. S. cerevisiae gDNA induces moderate Treg responses with restrained effector T cell expansion and higher checkpoint gene expression, entirely consistent with its commensal nature. In contrast, C. neoformans gDNA elicits a strongly inflammatory profile, promoting Th1/Th17 cells and driving high cytokine production. Mechanistically, C. albicans and S. cerevisiae gDNA dampen DNA-sensing pathways and enhance immune checkpoint molecules that act as brakes against overactivation, while C. neoformans gDNA robustly activates innate sensing pathways with limited checkpoint induction. These species-specific signaling profiles reveal that fungal gDNA itself can influence whether the immune system adopts a tolerant or inflammatory response toward fungi. This discovery highlights fungal genomic DNA as a previously underappreciated regulator of host–fungus interactions, offering new insight into commensal persistence, pathogenic invasion, and the potential for DNA-based antifungal interventions.