BosR (BB0647) Controls the RpoN-RpoS Regulatory Pathway and Virulence Expression in Borrelia burgdorferi by a Novel DNA-Binding Mechanism

Abstract

In Borrelia burgdorferi (Bb), the Lyme disease spirochete, the alternative σ factor σ54 (RpoN) directly activates transcription of another alternative σ factor, σS (RpoS) which, in turn, controls the expression of virulence-associated membrane lipoproteins. As is customary in σ54-dependent gene control, a putative NtrC-like enhancer-binding protein, Rrp2, is required to activate the RpoN-RpoS pathway. However, recently it was found that rpoS transcription in Bb also requires another regulator, BosR, which was previously designated as a Fur or PerR homolog. Given this unexpected requirement for a second activator to promote σ54-dependent gene transcription, and the fact that regulatory mechanisms among similar species of pathogenic bacteria can be strain-specific, we sought to confirm the regulatory role of BosR in a second virulent strain (strain 297) of Bb. Indeed, BosR displayed the same influence over lipoprotein expression and mammalian infectivity for strain Bb 297 that were previously noted for Bb strain B31. We subsequently found that recombinant BosR (rBosR) bound to the rpoS gene at three distinct sites, and that binding occurred despite the absence of consensus Fur or Per boxes. This led to the identification of a novel direct repeat sequence (TAAATTAAAT) critical for rBosR binding in vitro. Mutations in the repeat sequence markedly inhibited or abolished rBosR binding. Taken together, our studies provide new mechanistic insights into how BosR likely acts directly on rpoS as a positive transcriptional activator. Additional novelty is engendered by the facts that, although BosR is a Fur or PerR homolog and it contains zinc (like Fur and PerR), it has other unique features that clearly set it apart from these other regulators. Our findings also have broader implications regarding a previously unappreciated layer of control that can be involved in σ54–dependent gene regulation in bacteria.

The Fur homologue BosR requires Arg39 to activate rpoS transcription in Borrelia burgdorferi and thereby direct spirochaete infection in mice

“…In previous studies, knockouts of bosR were generated in strains B31-MI (Ouyang et al, 2009), ML23 (Hyde et al, 2009) and 297 (Ouyang et al, 2011). Strains B31-MI and 297 are infectious in mice (Ouyang et al, 2009(Ouyang et al, , 2011; however, strain ML23 lacks the lp25 linear plasmid and therefore is not infectious in mice (Hyde et al, 2009).…”

“…Strains B31-MI and 297 are infectious in mice (Ouyang et al, 2009(Ouyang et al, , 2011; however, strain ML23 lacks the lp25 linear plasmid and therefore is not infectious in mice (Hyde et al, 2009). Here, strain B31-A3 was chosen for study.…”

“…However, even though Rrp2 has a predicted DNA-binding domain, it appears that Rrp2 acts on RpoN without the benefit of binding DNA (Blevins et al, 2009;Burtnick et al, 2007;Yang et al, 2003). In vitro, BosR binds multiple sites within the rpoS promoter (Ouyang et al, 2011(Ouyang et al, , 2014(Ouyang et al, , 2015. Presumably, in vivo, BosR must bind one or more of these sites to activate rpoS transcription.…”

“…Although BosR may be involved in the oxidative stress response (Hyde et al, 2009(Hyde et al, , 2010), it appears its main function is to act as a global regulator. To prepare the spirochaete for mammalian infection, BosR upregulates transcription of rpoS (Hyde et al, 2009(Hyde et al, , 2010Ouyang et al, 2009Ouyang et al, , 2011 and also downregulates transcription of various tick-phase genes (e.g. ospA and ospD), which need only be expressed in the tick and which if allowed expression in the mammal could lead to immune clearance (Shi et al, 2014;Wang et al, 2013).…”

“…ospA and ospD), which need only be expressed in the tick and which if allowed expression in the mammal could lead to immune clearance (Shi et al, 2014;Wang et al, 2013). When BosR activates rpoS, it binds the direct repeat (DR) sequence TAAATT-AAAT (Ouyang et al, 2011); however, when BosR represses ospA and ospD, it binds a variant of the Per box sequence TTATAAT-ATTATAA (Wang et al, 2013). How BosR can bind two very different sequences and generate two very different outcomes is currently unclear.…”