The Borrelia burgdorferi RelA/SpoT Homolog and Stringent Response Regulate Survival in the Tick Vector and Global Gene Expression during Starvation

As the Lyme disease bacterium Borrelia burgdorferi traverses its enzootic cycle, alternating between a tick vector and a vertebrate host, the spirochete must adapt and persist in the tick midgut under prolonged nutrient stress between blood meals. In this study, we examined the role of the stringent response in tick persistence and in regulation of gene expression during nutrient limitation. Nutritionally starving B. burgdorferi in vitro increased the levels of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively referred to as (p)ppGpp, products of the bifunctional synthetase/hydrolase Rel(Bbu) (RelA/SpoT homolog). Conversely, returning B. burgdorferi to a nutrient-rich medium decreased (p)ppGpp levels. B. burgdorferi survival in ticks between the larval and nymph blood meals, and during starvation in vitro, was dependent on Rel(Bbu). Furthermore, normal morphological conversion from a flat-wave shape to a condensed round body (RB) form during starvation was dependent on Rel(Bbu); rel (Bbu) mutants more frequently formed RBs, but their membranes were compromised. By differential RNA sequencing analyses, we found that Rel(Bbu) regulates an extensive transcriptome, both dependent and independent of nutrient stress. The Rel(Bbu) regulon includes the glp operon, which is important for glycerol utilization and persistence in the tick, virulence factors and the late phage operon of the 32-kb circular plasmid (cp32) family. In summary, our data suggest that Rel(Bbu) globally modulates transcription in response to nutrient stress by increasing (p)ppGpp levels to facilitate B. burgdorferi persistence in the tick.