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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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Related in: MedlinePlus

A stable HrpRS hetero-hexameric species appears to be the active form.(a) Analytical HPLC gel-filtration vertically offset chromatograms of co-purified HrpRS (top); re-chromatographed fraction 7 (Hex re-run) and fraction 10 (monomer/dimer (M/d) re-run). The apparent monomer/dimer (m/d), hexamer (hex), void volume (void) retentions are indicated. Molecular mass standards (right) and their retention volumes used to calculate the apparent MW of the peaks. (b) SDS–PAGE of the SYPRO-stained fractions collected during gel-filtration in a. The relative fluorescence intensity ratios of HrpR/HrpS are plotted in the line graph (superimposed on the gel-filtration profile of HrpRS; grey). (c) Bar chart illustrating the ATPase turnover rates (min−1) of the fractions in b (superimposed on the gel-filtration profile of HrpRS; grey). Concentration-independent ATP turnover rates in a twofold dilution series of fraction 7 (hex) compared with dilutions of fraction 10 (m/d). (d) Glutaraldehyde crosslinked HrpR, HrpRS and PspFR168A complexes were separated by SDS–PAGE and analysed by western blotting using antibodies specific to HrpR (α-HrpR), HrpS (α-His) or PspF (α-PspF). Migration mobilities of several higher-order oligomeric species (labelled dimer, tetramer and hexamer) are indicated. (e) In vitro transcription assay (as in Fig. 2a, where FL represents the full-length transcript of ∼470 nt) of the monomer/dimer fraction 10 from a (m/d) at 0.2 μM compared with an equimolar concentration of HrpRS (0.2 μM), before gel filtration. In c, all assays were minimally performed in triplicates and standard errors of the mean are shown.
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f5: A stable HrpRS hetero-hexameric species appears to be the active form.(a) Analytical HPLC gel-filtration vertically offset chromatograms of co-purified HrpRS (top); re-chromatographed fraction 7 (Hex re-run) and fraction 10 (monomer/dimer (M/d) re-run). The apparent monomer/dimer (m/d), hexamer (hex), void volume (void) retentions are indicated. Molecular mass standards (right) and their retention volumes used to calculate the apparent MW of the peaks. (b) SDS–PAGE of the SYPRO-stained fractions collected during gel-filtration in a. The relative fluorescence intensity ratios of HrpR/HrpS are plotted in the line graph (superimposed on the gel-filtration profile of HrpRS; grey). (c) Bar chart illustrating the ATPase turnover rates (min−1) of the fractions in b (superimposed on the gel-filtration profile of HrpRS; grey). Concentration-independent ATP turnover rates in a twofold dilution series of fraction 7 (hex) compared with dilutions of fraction 10 (m/d). (d) Glutaraldehyde crosslinked HrpR, HrpRS and PspFR168A complexes were separated by SDS–PAGE and analysed by western blotting using antibodies specific to HrpR (α-HrpR), HrpS (α-His) or PspF (α-PspF). Migration mobilities of several higher-order oligomeric species (labelled dimer, tetramer and hexamer) are indicated. (e) In vitro transcription assay (as in Fig. 2a, where FL represents the full-length transcript of ∼470 nt) of the monomer/dimer fraction 10 from a (m/d) at 0.2 μM compared with an equimolar concentration of HrpRS (0.2 μM), before gel filtration. In c, all assays were minimally performed in triplicates and standard errors of the mean are shown.

Mentions: Gel-filtration of co-purified HrpRS (Fig. 5a) resolved at least two distinct species with molecular weights (MWs) corresponding to 212 kDa (apparent 6.1 mer) and 52 kDa (apparent 1.4 mer)—where the MW of HrpR is 34.9 kDa and HrpSHis is 34.5 kDa. The lower MW peak does not permit assignment of subunit composition(s) and may comprise a mixture of monomers and dimers. SDS–PAGE analysis (Fig. 5b) of the fractions demonstrates that HrpR and HrpS are present in both peaks. Interestingly, we note that the relative intensities of HrpR and HrpS differ significantly between the hexameric (hex) species (where HrpS predominates over HrpR) and monomer/dimer species (where HrpR predominates over HrpS). The relative HrpR/HrpS fluorescence intensities are consistently scored as ∼0.5 in fractions 5–8; suggesting a fixed HrpR/HrpS stoichiometry in the hexameric fractions—although precise determination of the stoichiometry of this complex was not possible using this approach. Re-produced retention volumes (Fig. 5a, hex re-run) and concentration-independent ATPase turnover rates of the re-chromatographed or diluted hexameric HrpRS fraction (Fig. 5c; fraction 7) indicate that the HrpRS hexamer is stable minimally over the purification and assay time course and at the concentrations used. Further evidence that HrpRS forms a mixed subunit hexameric assembly was obtained using glutaraldehyde protein–protein crosslinking (Fig. 5d); where a hexameric crosslinked species that reacted to HrpR- (α-HrpR) and His-tag-antibodies (to detect HrpS; α-His) was detected.


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)

A stable HrpRS hetero-hexameric species appears to be the active form.(a) Analytical HPLC gel-filtration vertically offset chromatograms of co-purified HrpRS (top); re-chromatographed fraction 7 (Hex re-run) and fraction 10 (monomer/dimer (M/d) re-run). The apparent monomer/dimer (m/d), hexamer (hex), void volume (void) retentions are indicated. Molecular mass standards (right) and their retention volumes used to calculate the apparent MW of the peaks. (b) SDS–PAGE of the SYPRO-stained fractions collected during gel-filtration in a. The relative fluorescence intensity ratios of HrpR/HrpS are plotted in the line graph (superimposed on the gel-filtration profile of HrpRS; grey). (c) Bar chart illustrating the ATPase turnover rates (min−1) of the fractions in b (superimposed on the gel-filtration profile of HrpRS; grey). Concentration-independent ATP turnover rates in a twofold dilution series of fraction 7 (hex) compared with dilutions of fraction 10 (m/d). (d) Glutaraldehyde crosslinked HrpR, HrpRS and PspFR168A complexes were separated by SDS–PAGE and analysed by western blotting using antibodies specific to HrpR (α-HrpR), HrpS (α-His) or PspF (α-PspF). Migration mobilities of several higher-order oligomeric species (labelled dimer, tetramer and hexamer) are indicated. (e) In vitro transcription assay (as in Fig. 2a, where FL represents the full-length transcript of ∼470 nt) of the monomer/dimer fraction 10 from a (m/d) at 0.2 μM compared with an equimolar concentration of HrpRS (0.2 μM), before gel filtration. In c, all assays were minimally performed in triplicates and standard errors of the mean are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: A stable HrpRS hetero-hexameric species appears to be the active form.(a) Analytical HPLC gel-filtration vertically offset chromatograms of co-purified HrpRS (top); re-chromatographed fraction 7 (Hex re-run) and fraction 10 (monomer/dimer (M/d) re-run). The apparent monomer/dimer (m/d), hexamer (hex), void volume (void) retentions are indicated. Molecular mass standards (right) and their retention volumes used to calculate the apparent MW of the peaks. (b) SDS–PAGE of the SYPRO-stained fractions collected during gel-filtration in a. The relative fluorescence intensity ratios of HrpR/HrpS are plotted in the line graph (superimposed on the gel-filtration profile of HrpRS; grey). (c) Bar chart illustrating the ATPase turnover rates (min−1) of the fractions in b (superimposed on the gel-filtration profile of HrpRS; grey). Concentration-independent ATP turnover rates in a twofold dilution series of fraction 7 (hex) compared with dilutions of fraction 10 (m/d). (d) Glutaraldehyde crosslinked HrpR, HrpRS and PspFR168A complexes were separated by SDS–PAGE and analysed by western blotting using antibodies specific to HrpR (α-HrpR), HrpS (α-His) or PspF (α-PspF). Migration mobilities of several higher-order oligomeric species (labelled dimer, tetramer and hexamer) are indicated. (e) In vitro transcription assay (as in Fig. 2a, where FL represents the full-length transcript of ∼470 nt) of the monomer/dimer fraction 10 from a (m/d) at 0.2 μM compared with an equimolar concentration of HrpRS (0.2 μM), before gel filtration. In c, all assays were minimally performed in triplicates and standard errors of the mean are shown.
Mentions: Gel-filtration of co-purified HrpRS (Fig. 5a) resolved at least two distinct species with molecular weights (MWs) corresponding to 212 kDa (apparent 6.1 mer) and 52 kDa (apparent 1.4 mer)—where the MW of HrpR is 34.9 kDa and HrpSHis is 34.5 kDa. The lower MW peak does not permit assignment of subunit composition(s) and may comprise a mixture of monomers and dimers. SDS–PAGE analysis (Fig. 5b) of the fractions demonstrates that HrpR and HrpS are present in both peaks. Interestingly, we note that the relative intensities of HrpR and HrpS differ significantly between the hexameric (hex) species (where HrpS predominates over HrpR) and monomer/dimer species (where HrpR predominates over HrpS). The relative HrpR/HrpS fluorescence intensities are consistently scored as ∼0.5 in fractions 5–8; suggesting a fixed HrpR/HrpS stoichiometry in the hexameric fractions—although precise determination of the stoichiometry of this complex was not possible using this approach. Re-produced retention volumes (Fig. 5a, hex re-run) and concentration-independent ATPase turnover rates of the re-chromatographed or diluted hexameric HrpRS fraction (Fig. 5c; fraction 7) indicate that the HrpRS hexamer is stable minimally over the purification and assay time course and at the concentrations used. Further evidence that HrpRS forms a mixed subunit hexameric assembly was obtained using glutaraldehyde protein–protein crosslinking (Fig. 5d); where a hexameric crosslinked species that reacted to HrpR- (α-HrpR) and His-tag-antibodies (to detect HrpS; α-His) was detected.

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