Rrp2, a Prokaryotic Enhancer-Like Binding Protein, Is Essential for Viability of Borrelia burgdorferi

Abstract

The Lyme disease spirochete, Borrelia burgdorferi, exists in two diverse niches (i.e., an arthropod tick vector and mammalian host) during its enzootic life cycle. To effectively adapt to these unique environments, the bacterium alters the expression of numerous genes, including several major outer surface (lipo)proteins that are required for infection and transmission. An enhancerbinding protein (EBP), known as Rrp2, is one identified activator of the RpoN/RpoS alternative sigma factor cascade. Because initial efforts to generate an rrp2 deletion strain were unsuccessful, the role of Rrp2 in the activation of the RpoN/RpoS pathway was first defined using a strain of B. burgdorferi carrying an rrp2 point mutant that was defective in its ability to activate RpoNdependent transcription. The fact that subsequent attempts to disrupt rrp2 have also been unsuccessful has led investigators to hypothesize that Rrp2 has other undefined functions which are essential for B. burgdorferi survival and independent of its EBP function. We used a lac-based inducible expression system to generate a conditional rrp2 mutant in virulent B. burgdorferi. In this strain, an isopropyl-␤-D-thiogalactopyranoside-inducible copy of the rrp2 gene is expressed in trans from a borrelial shuttle vector. We found that the chromosomal copy of rrp2 could be inactivated only when rrp2 was induced, and the maintenance of rrp2 expression was required for the growth of the mutants. In addition, the overexpression of rrp2 is detrimental to B. burgdorferi growth in a manner that is independent of the RpoN/RpoS pathway. These studies provide the first direct evidence that rrp2 is an essential gene in B. burgdorferi.

BB0238, a Presumed Tetratricopeptide Repeat-Containing Protein, Is Required during Borrelia burgdorferi Mammalian Infection

“…To ligate the two flanking regions, the downstream F2 fragment was excised using BssHII and ligated into the vector containing the upstream F1 region linearized with AscI. A Kan r marker with a T7 transcriptional terminator fused to the end of the resistance gene, designated flgBp-aphI-T7t, was provided by Scott Samuels at the University of Montana (17). The Kan r marker was ligated into the unique AscI restriction site at the junction between the two flanking regions.…”

“…The rrp2 ORF in piRrp2(Kan) (17) was then replaced by the ebfC ORF to generate pJSB499. pJSB499 was transformed into Bb297, and Kan r transformants were recovered.…”

Insight into the Dual Functions of Bacterial Enhancer-Binding Protein Rrp2 of Borrelia burgdorferi

“…burgdorferi growth (25). Unlike rpoN or rpoS, which can readily be inactivated in B. burgdorferi, attempts by multiple groups to generate an rrp2 deletion mutant have failed (7,22,23).…”

“…Unlike rpoN or rpoS, which can readily be inactivated in B. burgdorferi, attempts by multiple groups to generate an rrp2 deletion mutant have failed (7,22,23). Recently, a conditional rrp2 mutant was constructed in the presence of an isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible copy of rrp2 (25). The conditional rrp2 mutant was unable to replicate in the absence of IPTG, demonstrating that Rrp2 is indispensable for borrelial growth.…”

Construction and Characterization of a Borrelia burgdorferi Strain with Conditional Expression of the Essential Telomere Resolvase, ResT

“…However, the recent development of inducible expression systems in B. burgdorferi has provided a powerful new approach in the arsenal of genetic tools available (38)(39)(40). In particular, the expression vector pJSB104 (39), in which the expressed gene is regulated by a double lac operator and the lac repressor, has allowed for tight regulation and the generation of conditional mutants in two essential genes: the dedA orthologue bb0250 (41) and the gene for the response regulator protein, rrp2 (42). Here we report the use of the pJBS104 expression system to generate a conditional mutation in the essential B. burgdorferi telomere resolvase resT.…”