The diguanylate cyclase, Rrp1, regulates critical steps in the enzootic cycle of the Lyme disease spirochetes.

Rrp1 is the sole c-di-GMP-producing protein (diguanylate cyclase) of Borrelia burgdorferi. To test the hypothesis that Rrp1 regulates critical processes involved in the transmission of spirochetes between ticks and mammals, an rrp1 deletion mutant (B31-Δrrp1) and a strain that constitutively produces elevated levels of Rrp1 (B31-OV) were constructed. The strains were assessed for progression through the enzootic cycle using an Ixodes tick/C3H-HeJ mouse model and tick immersion feeding methods. B31-Δrrp1 infected mice as efficiently as wild type but had altered motility, decreased chemotactic responses to N-acetylglucosamine (NAG) and attenuated ability to disseminate or colonize distal organs. While this strain infected mice, it was not able to survive in ticks. In contrast, B31-OV displayed normal motility patterns and chemotactic responses but was non-infectious in mice. Using immersion feeding techniques, we demonstrate that B31-OV can establish a population in ticks and survive exposure to a natural bloodmeal. The results presented here indicate Rrp1, and by extension, c-di-GMP, are not strictly required for murine infection, but are required for the successful establishment of a productive population of B. burgdorferi in ticks. These analyses provide significant new insight into the genetic regulatory mechanisms of the Lyme disease spirochetes.

Identification and molecular characterization of a cyclic-di-GMP effector protein, PlzA (BB0733): additional evidence for the existence of a functional cyclic-di-GMP regulatory network in the Lyme disease spirochete, Borrelia burgdorferi.

The Borrelia burgdorferi Rrp1 protein is a diguanylate cyclase that controls a regulon consisting of approximately 10% of the total genome. Because Rrp1 lacks a DNA-binding domain, its regulatory capability is most likely mediated through the production of bis-(3'-5')-cyclic dimeric GMP (c-di-GMP). C-di-GMP binds to and activates the regulatory activity of proteins that harbor a PilZ domain. The occurrence of a PilZ domain within a protein is not in and of itself sufficient to convey c-di-GMP binding, as other structural aspects of the protein are important in the interaction. In this study, we have assessed the expression and c-di-GMP binding ability of the sole PilZ domain-containing protein of B. burgdorferi B31, PlzA. PlzA was determined to be upregulated by tick feeding and to be expressed during mammalian infection. The gene is highly conserved and present in all Borrelia species. Analyses of recombinant PlzA demonstrated its ability to bind c-di-GMP and site-directed mutagenesis revealed that this interaction is highly specific and dependent on Arg residues contained within the PilZ domain. In summary, this study is the first to identify a c-di-GMP effector molecule in a spirochete and provides additional evidence for the existence of a complete c-di-GMP regulatory network in the Lyme disease spirochete, B. burgdorferi.