Two Distinct Mechanisms Govern RpoS-Mediated Repression of Tick-Phase Genes during Mammalian Host Adaptation by
Borrelia burgdorferi
, the Lyme Disease Spirochete
Authors: Grove Arianna P. , Liveris Dionysios , Iyer Radha , Petzke Mary , Rudman Joseph , Caimano Melissa J. , Radolf Justin D. , Schwartz Ira 
Published: August 2017

The alternative sigma factor RpoS plays a key role modulating gene expression in
Borrelia burgdorferi
, the Lyme disease spirochete, by transcribing mammalian host-phase genes and repressing σ
70
-dependent genes required within the arthropod vector. To identify
cis
regulatory elements involved in RpoS-dependent repression, we analyzed green fluorescent protein (GFP) transcriptional reporters containing portions of the upstream regions of the prototypical tick-phase genes
ospAB
, the
glp
operon, and
bba74
. As RpoS-mediated repression occurs only following mammalian host adaptation, strains containing the reporters were grown in dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats. Wild-type spirochetes harboring
ospAB
- and
glp-gfp
constructs containing only the minimal (−35/−10) σ
70
promoter elements had significantly lower expression in DMCs relative to growth
in vitro
at 37°C; no reduction in expression occurred in a DMC-cultivated RpoS mutant harboring these constructs. In contrast, RpoS-mediated repression of
bba74
required a stretch of DNA located between −165 and −82 relative to its transcriptional start site. Electrophoretic mobility shift assays employing extracts of DMC-cultivated
B. burgdorferi
produced a gel shift, whereas extracts from RpoS mutant spirochetes did not. Collectively, these data demonstrate that RpoS-mediated repression of tick-phase borrelial genes occurs by at least two distinct mechanisms. One (e.g.,
ospAB
and the
glp
operon) involves primarily sequence elements near the core promoter, while the other (e.g.,
bba74
) involves an RpoS-induced transacting repressor. Our results provide a genetic framework for further dissection of the essential “gatekeeper” role of RpoS throughout the
B. burgdorferi
enzootic cycle.

IMPORTANCE
Borrelia burgdorferi
, the Lyme disease spirochete, modulates gene expression to adapt to the distinctive environments of its mammalian host and arthropod vector during its enzootic cycle. The alternative sigma factor RpoS has been referred to as a “gatekeeper” due to its central role in regulating the reciprocal expression of mammalian host- and tick-phase genes. While RpoS-dependent transcription has been studied extensively, little is known regarding the mechanism(s) of RpoS-mediated repression. We employed a combination of green fluorescent protein transcriptional reporters along with an
in vivo
model to define
cis
regulatory sequences responsible for RpoS-mediated repression of prototypical tick-phase genes. Repression of
ospAB
and the
glp
operon requires only sequences near their core promoters, whereas modulation of
bba74
expression involves a putative RpoS-dependent repressor that binds upstream of the core promoter. Thus, Lyme disease spirochetes employ at least two different RpoS-dependent mechanisms to repress tick-phase genes within the mammal.