Assessing the Potential for DNA Quadruplex Formation in the Predatory Bacterium Bdellovibrio bacteriovorus

[Image: see text] During its life cycle, the predatory bacterium Bdellovibrio bacteriovorus switches between an attack and a growth phase, each of which is characterized by a distinct pattern of gene expression. Twenty-one potential G-quadruplex-forming sequences (PQFS) have been identified in the Bdellovibrio genome. These G-rich sequences are prevalent within open reading frames and nearly evenly distributed between the template and the coding strand, suggesting that they could play a role in gene expression and life cycle switching. Published transcriptomic data show that the genes nearest these sequences are not (de)activated together during the same phases of the life cycle. We explored the biophysical properties of three identified PQFS using circular dichroism (CD) spectroscopy and gel electrophoresis and demonstrated that all three sequences fold into stable unimolecular quadruplexes with distinct topologies. In the presence of their complementary strands, each forms an equilibrium mixture of duplex and quadruplex in which quadruplex formation is favored at higher temperatures. Once the quadruplexes are folded, they are slow to form a duplex when the complementary strand is added, with one sequence requiring the equivalent of many Bdellovibrio lifetimes to do so. Using a variety of cosolutes, we showed that molecular crowding mimicking cellular conditions stabilizes the quadruplex structures and induces structural transitions to the parallel topology regardless of the original topology. Taken together, these experiments suggest that Bdellovibrio PQFS are capable of forming quadruplexes in vivo and thereby playing a role in gene expression.