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

Jovanovic M, James EH, Burrows PC, Rego FG, Buck M, Schumacher J - Nat Commun (2011)

Bottom Line: 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 σ(54)-RNA polymerase activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Faculty of Natural Sciences, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
The bacterial AAA+ enhancer-binding proteins (EBPs) HrpR and HrpS (HrpRS) of Pseudomonas syringae (Ps) activate σ(54)-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 σ(54)-RNA polymerase activation.

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ATP binding to HrpR and HrpS subunits.SDS–PAGE gel of ultraviolet radiated proteins. (a) Co-purified HrpRS in the presence of [α-32P]ATP and when supplemented with excess of non-labelled competitor nucleotides (as indicated). The relative concentrations of the Hrp proteins were visualized using SYPRO Ruby protein staining (Sy), and were compared with the relative intensities of the crosslinked radioactive species (determined by PhosphoImager analysis; IP). Full gel images are shown in Supplementary Figure 5. (b) As in a using PspF1−275 as comparative control, for which specific ATP-binding methodology has been established3945.
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f4: ATP binding to HrpR and HrpS subunits.SDS–PAGE gel of ultraviolet radiated proteins. (a) Co-purified HrpRS in the presence of [α-32P]ATP and when supplemented with excess of non-labelled competitor nucleotides (as indicated). The relative concentrations of the Hrp proteins were visualized using SYPRO Ruby protein staining (Sy), and were compared with the relative intensities of the crosslinked radioactive species (determined by PhosphoImager analysis; IP). Full gel images are shown in Supplementary Figure 5. (b) As in a using PspF1−275 as comparative control, for which specific ATP-binding methodology has been established3945.

Mentions: Identical subunits in EBPs and other AAA+ proteins make delineation of mechanistic roles of individual subunits experimentally challenging44. To directly probe any non-equivalence of HrpR and HrpS subunits (as suggested by the mutagenesis studies; Fig. 1c,d), in the context of the purified functional HrpRS complex, we tested nucleotide binding with non-equilibrium covalent ultraviolet crosslinking3945 of [α-32P]ATP using PspF as a control. Recall that the highly conserved Walker A motif (GXXXXGK[T/S]), especially, the 'K' residue, is strictly required for nucleotide binding in AAA+ proteins46, including EBPs3947. As shown in Figure 4, HrpR and HrpS were separated by SDS–PAGE and the relative protein concentrations evaluated by SYPRO ruby protein stain fluorescence (Sy). The relative amount of [α-32P]ATP crosslinked protein species was determined by PhosphorImager analysis. Side-by-side comparison of the relative [α-32P]ATP/fluorescence intensities (Fig. 4a) of the HrpR and HrpS bands indicate that [α-32P]ATP preferentially crosslinks to HrpR and at a level comparable to that of the structurally and functionally characterized EBP PspF39 (Fig. 4b). Specific binding of [α-32P]ATP to HrpR is further demonstrated by the competitive binding of non-radiolabelled ADP, ATP and ATPγS (Fig. 4a). Differential nucleotide binding to particular subunits of the strictly co-dependent HrpRS system provides direct evidence for non-equivalent nucleotide-dependent roles for individual subunits in a functional EBP complex.


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

Jovanovic M, James EH, Burrows PC, Rego FG, Buck M, Schumacher J - Nat Commun (2011)

ATP binding to HrpR and HrpS subunits.SDS–PAGE gel of ultraviolet radiated proteins. (a) Co-purified HrpRS in the presence of [α-32P]ATP and when supplemented with excess of non-labelled competitor nucleotides (as indicated). The relative concentrations of the Hrp proteins were visualized using SYPRO Ruby protein staining (Sy), and were compared with the relative intensities of the crosslinked radioactive species (determined by PhosphoImager analysis; IP). Full gel images are shown in Supplementary Figure 5. (b) As in a using PspF1−275 as comparative control, for which specific ATP-binding methodology has been established3945.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105312&req=5

f4: ATP binding to HrpR and HrpS subunits.SDS–PAGE gel of ultraviolet radiated proteins. (a) Co-purified HrpRS in the presence of [α-32P]ATP and when supplemented with excess of non-labelled competitor nucleotides (as indicated). The relative concentrations of the Hrp proteins were visualized using SYPRO Ruby protein staining (Sy), and were compared with the relative intensities of the crosslinked radioactive species (determined by PhosphoImager analysis; IP). Full gel images are shown in Supplementary Figure 5. (b) As in a using PspF1−275 as comparative control, for which specific ATP-binding methodology has been established3945.
Mentions: Identical subunits in EBPs and other AAA+ proteins make delineation of mechanistic roles of individual subunits experimentally challenging44. To directly probe any non-equivalence of HrpR and HrpS subunits (as suggested by the mutagenesis studies; Fig. 1c,d), in the context of the purified functional HrpRS complex, we tested nucleotide binding with non-equilibrium covalent ultraviolet crosslinking3945 of [α-32P]ATP using PspF as a control. Recall that the highly conserved Walker A motif (GXXXXGK[T/S]), especially, the 'K' residue, is strictly required for nucleotide binding in AAA+ proteins46, including EBPs3947. As shown in Figure 4, HrpR and HrpS were separated by SDS–PAGE and the relative protein concentrations evaluated by SYPRO ruby protein stain fluorescence (Sy). The relative amount of [α-32P]ATP crosslinked protein species was determined by PhosphorImager analysis. Side-by-side comparison of the relative [α-32P]ATP/fluorescence intensities (Fig. 4a) of the HrpR and HrpS bands indicate that [α-32P]ATP preferentially crosslinks to HrpR and at a level comparable to that of the structurally and functionally characterized EBP PspF39 (Fig. 4b). Specific binding of [α-32P]ATP to HrpR is further demonstrated by the competitive binding of non-radiolabelled ADP, ATP and ATPγS (Fig. 4a). Differential nucleotide binding to particular subunits of the strictly co-dependent HrpRS system provides direct evidence for non-equivalent nucleotide-dependent roles for individual subunits in a functional EBP complex.

Bottom Line: 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 σ(54)-RNA polymerase activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Faculty of Natural Sciences, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK.

ABSTRACT
The bacterial AAA+ enhancer-binding proteins (EBPs) HrpR and HrpS (HrpRS) of Pseudomonas syringae (Ps) activate σ(54)-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 σ(54)-RNA polymerase activation.

Show MeSH
Related in: MedlinePlus