The putative Walker A and Walker B motifs of Rrp2 are required for the growth of Borrelia burgdorferi

Abstract

SummaryRrp2 encodes a putative bacterial enhancer binding protein (bEBP) in Borrelia burgdorferi. Point mutation (G239C) of Rrp2 abolishes the transcriptional activation of r 54 -dependent rpoS. In contrast to canonical bEBPs that are dispensable for bacterial growth, Rrp2 is essential for borrelial growth in BSK medium. It has been believed that Rrp2's ATPase activity is not required for cell growth, but experimental evidence supporting this notion has been lacking. In particular, it has remained unclear whether the residue G239 is involved in Rrp2's presumptive ATPase activity. To address these information gaps, we examined the roles of Rrp2's potential strategic signatures including the G239 residue and the putative Walker A and Walker B motifs. Herein it was showed that Rrp2 has ATP binding and hydrolysis activities engendered by the Walker A and B motifs respectively. However, these activities were not significantly impaired by a G239C mutation. Further mutagenesis analyses indicated that Rrp2's Walker A and B motifs are required for borrelial growth; mutations of key residues in these two motifs were lethal to B. burgdorferi. The combined data suggest that the Walker A and Walker B motifs of Rrp2 are involved in the control of another unknown RpoS-independent gene product(s) associated with borrelial replication.

The oligopeptide ABC transporter OppA4 negatively regulates the virulence factor OspC production of the Lyme disease pathogen

“…We did not examine the level of rrp2 and rpoN in the oppA4 mutant, as activation of rpoS by Rrp2 and RpoN is controlled by phosphorylation of Rrp2. We and others recently showed that under the in vitro cultivation conditions, Rrp2 phosphorylation appears to be constitutive, as Rrp2 phosphorylation is vital for B. burgdorferi replication (Groshong et al, 2012; Ouyang and Zhou, 2016; Yin et al, 2016). How OppA4 negatively regulates bosR expression warrants further investigation.…”

The Emerging Role of Microbial Biofilm in Lyme Neuroborreliosis

“…The RpoN–RpoS pathway relies on the activity of RpoN (σ 54 ), which controls the transcription of RpoS (σ S ) through the binding to a canonical −24/−12 RpoN-type promoter sequence (95, 193). The activation of the σ 54 -σ S cascade, in turn, is modulated by a bacterial enhancer-binding protein (bEBP)/σ54-dependent activator (Rrp2) in concert with BosR (91, 95, 193–198). After the activation, RpoS acts as a global gene regulator controlling the expression of over 100 different genes involved in stress responses, host infection and survival, including biofilm formation (87, 95, 182).…”

Gene Regulation and Transcriptomics

“…Yang et al (2003) first demonstrated that Rrp2 is the upstream activator for the RpoN-RpoS alternative σ factor cascade (Figure 2): a single G239C point mutation within the central activation domain of Rrp2 abolished expression of rpoS, ospC, and other RpoN-RpoS pathway-dependent genes. The G239C mutation did not abolish the ATPase activity of Rrp2, suggesting that the mutation affects interaction of Rrp2 with RpoN necessary for initiation of open complex formation (Ouyang and Zhou, 2017). Artificial induction of an inducible rrp2 construct also resulted in rpoS transcription (Ouyang et al, 2014c).…”

“…This suggests that Rrp2 has an additional role besides serving as the activator for the RpoN-RpoS σ factor cascade. Phosphorylation, ATPase activity, and the C-terminal-mediated DNA-binding are all required for the essential function of Rrp2, but the mechanistic details remain unclear (Yin et al, 2016;Ouyang and Zhou, 2017). Further work, such as chromatin immunoprecipitation (ChIP), will be required to identify Rrp2-dependent but RpoNindependent genes to gain insight the essential nature of Rrp2.…”