Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity

Abstract

The bacterial AAA+ enhancer-binding proteins (EBPs) HrpR and HrpS (HrpRS) of Pseudomonas syringae (Ps) activate σ54-dependent transcription at the hrpL promoter; triggering type-three secretion system-mediated pathogenicity. In contrast with singly acting EBPs, the evolution of the strictly co-operative HrpRS pair raises questions of potential benefits and mechanistic differences this transcription control system offers. Here, we show distinct properties of HrpR and HrpS variants, indicating functional specialization of these non-redundant, tandemly arranged paralogues. Activities of HrpR, HrpS and their control proteins HrpV and HrpG from Ps pv. tomato DC3000 in vitro establish that HrpRS forms a transcriptionally active hetero-hexamer, that there is a direct negative regulatory role for HrpV through specific binding to HrpS and that HrpG suppresses HrpV. The distinct HrpR and HrpS functionalities suggest how partial paralogue degeneration has potentially led to a novel control mechanism for EBPs and indicate subunit-specific roles for EBPs in σ54-RNA polymerase activation.

Negative Autogenous Control of the Master Type III Secretion System Regulator HrpL inPseudomonas syringae

“…E. coli has been utilized previously as a heterologous model system in which to study the initiation of hrpL transcription independently of the wider DC3000-specific regulatory network (15). In this study, a multiple plasmid-based system was engineered in the E. coli s17λpir strain with IPTG (isopropyl-β- d -thiogalactopyranoside)-induced heterologous expression of hrpRS from pAPT- hrpRS , driving activation of the pBBR1-P hrpL - gfp reporter.…”

“…Therefore, a trinucleotide GGA>AAC substitution was introduced in the pBBR1- rfp -P hrpL (Δ35e)-g fp reporter to inhibit the association of HrpL at this site. Finally, a further reporter construct was generated consisting of the minimal sufficient P hrpL promoter sequence (−147 to +1) (15) but lacking the entire P hrpJ promoter, pBBR1-P hrpL (147)- gfp . Together with the pSEVA614- hrpL and pSEVA614- hrpL Δ4.2 expression plasmids, this series of reporters were used to investigate the specific role of HrpL DNA-binding function at P hrpJ for NAC (Fig.…”

“…4b). This implies that P hrpJ -bound RNAP-HrpL occludes a significant region of P hrpL , including the predicted UAS for HrpRS binding (15) and part of the IHF recognition sequence. NAC might therefore be manifest via interference with HrpRS and/or IHF DNA binding by RNAP-HrpL.…”

“…Integration host factor (IHF)-mediated DNA looping facilitates contact between the promoter-bound EBP complex, usually homohexameric, and the inactive RNA polymerase (RNAP)-σ 54 complex. In P. syringae , transcription of hrpL is atypically activated by a heterohexamer comprising two codependent EBPs, HrpR and HrpS (15). Furthermore, HrpS is subjected to allosteric posttranslational inhibition by HrpV, which is in turn sequestered by HrpG (16).…”

The EbpA-RpoN Regulatory Pathway of the Pathogen Leptospira interrogans Is Essential for Survival in the Environment

“…RpoN has been shown to be involved in regulation of many bacterial functions such as nitrogen metabolism, flagellar biosynthesis, osmotolerance, pH changes, biofilm formation, motility, colonization, lipoprotein biosynthesis, and a type III secretion system (18,(27)(28)(29). Bioinformatics analysis predicted 13 putative RpoN ( 54 ) (32).…”