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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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hrpL promoter functional UAS site mapping and promoter binding.(a) Overview of upstream promoter truncation constructs of the hrpL promoter DNA::lacZ repoter gene (A: −600 to +58; B: −147 to +58; C: −101 to +58 and D: −36 to +58; numbering with respect to the transcription start-site, +1) used to map functional UAS sequences and showing candidate UAS upstream activator sequences (black boxes), IHF (integration host factor) and σ54-RNAP-binding sites. (b) In vivo transcription from reporter constructs shown in a, in either wild-type E.coli (MC4100, black bars) or an isogenic strain deleted for IHF coding sequence (grey bar). (c) Electrophoretic mobility shift assay using the hrpL promoter probe with increasing HrpR, HrpS, pre-mixed HrpR and HrpS (HrpR+HrpS) concentrations (as indicated). (d) As in c with co-expressed, co-purified HrpRS (HrpRS). (e) Electrophoretic mobility shift assay of the hrpL promoter probe with (10 nM) IHF. In b, all assays were minimally performed in triplicate and standard errors of the mean are shown.
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f3: hrpL promoter functional UAS site mapping and promoter binding.(a) Overview of upstream promoter truncation constructs of the hrpL promoter DNA::lacZ repoter gene (A: −600 to +58; B: −147 to +58; C: −101 to +58 and D: −36 to +58; numbering with respect to the transcription start-site, +1) used to map functional UAS sequences and showing candidate UAS upstream activator sequences (black boxes), IHF (integration host factor) and σ54-RNAP-binding sites. (b) In vivo transcription from reporter constructs shown in a, in either wild-type E.coli (MC4100, black bars) or an isogenic strain deleted for IHF coding sequence (grey bar). (c) Electrophoretic mobility shift assay using the hrpL promoter probe with increasing HrpR, HrpS, pre-mixed HrpR and HrpS (HrpR+HrpS) concentrations (as indicated). (d) As in c with co-expressed, co-purified HrpRS (HrpRS). (e) Electrophoretic mobility shift assay of the hrpL promoter probe with (10 nM) IHF. In b, all assays were minimally performed in triplicate and standard errors of the mean are shown.

Mentions: We established that the hrpL promoter (PhrpL) contains a functional UAS between positions −147 to −36 relative to the transcription start-site (Fig. 3a,b), to which purified HrpR, HrpS and HrpRS bind in electrophoretic mobility shift assays (Fig. 3c,d). Consistent with the in vitro observations, in which addition of IHF greatly stimulated transcription (Fig. 2a), hrpL-dependent in vivo transcription in an IHF deletion strain was marginal (Fig. 3b). The specific IHF-binding site is present in the promoter probe (Fig. 3e), probably promoting productive interactions between HrpRS and σ54-RNAP, as evidenced by the increased transcription activity in vivo (Fig. 3b) and in vitro (Fig. 2a). Compared with either HrpR alone or HrpS alone or when HrpR and HrpS were mixed together (denoted as HrpR+HrpS, Fig. 3c), co-purified HrpRS bound the UAS DNA with greater affinity (Fig. 3d), indicating co-operative binding to the PhrpL. Lack of increased UAS binding in the presence of pre-mixed, separately purified HrpR and HrpS, probably reflects incompetent subunit assembly (in line with the in vitro transcription assays; Fig. 2b). These features of the hrpL promoter are in good agreement with other EBP interactions at σ54-dependent promoters in terms of co-operative binding to the UAS and exclude promoter architecture as the major basis of HrpRS co-dependency43.


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)

hrpL promoter functional UAS site mapping and promoter binding.(a) Overview of upstream promoter truncation constructs of the hrpL promoter DNA::lacZ repoter gene (A: −600 to +58; B: −147 to +58; C: −101 to +58 and D: −36 to +58; numbering with respect to the transcription start-site, +1) used to map functional UAS sequences and showing candidate UAS upstream activator sequences (black boxes), IHF (integration host factor) and σ54-RNAP-binding sites. (b) In vivo transcription from reporter constructs shown in a, in either wild-type E.coli (MC4100, black bars) or an isogenic strain deleted for IHF coding sequence (grey bar). (c) Electrophoretic mobility shift assay using the hrpL promoter probe with increasing HrpR, HrpS, pre-mixed HrpR and HrpS (HrpR+HrpS) concentrations (as indicated). (d) As in c with co-expressed, co-purified HrpRS (HrpRS). (e) Electrophoretic mobility shift assay of the hrpL promoter probe with (10 nM) IHF. In b, all assays were minimally performed in triplicate and standard errors of the mean are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: hrpL promoter functional UAS site mapping and promoter binding.(a) Overview of upstream promoter truncation constructs of the hrpL promoter DNA::lacZ repoter gene (A: −600 to +58; B: −147 to +58; C: −101 to +58 and D: −36 to +58; numbering with respect to the transcription start-site, +1) used to map functional UAS sequences and showing candidate UAS upstream activator sequences (black boxes), IHF (integration host factor) and σ54-RNAP-binding sites. (b) In vivo transcription from reporter constructs shown in a, in either wild-type E.coli (MC4100, black bars) or an isogenic strain deleted for IHF coding sequence (grey bar). (c) Electrophoretic mobility shift assay using the hrpL promoter probe with increasing HrpR, HrpS, pre-mixed HrpR and HrpS (HrpR+HrpS) concentrations (as indicated). (d) As in c with co-expressed, co-purified HrpRS (HrpRS). (e) Electrophoretic mobility shift assay of the hrpL promoter probe with (10 nM) IHF. In b, all assays were minimally performed in triplicate and standard errors of the mean are shown.
Mentions: We established that the hrpL promoter (PhrpL) contains a functional UAS between positions −147 to −36 relative to the transcription start-site (Fig. 3a,b), to which purified HrpR, HrpS and HrpRS bind in electrophoretic mobility shift assays (Fig. 3c,d). Consistent with the in vitro observations, in which addition of IHF greatly stimulated transcription (Fig. 2a), hrpL-dependent in vivo transcription in an IHF deletion strain was marginal (Fig. 3b). The specific IHF-binding site is present in the promoter probe (Fig. 3e), probably promoting productive interactions between HrpRS and σ54-RNAP, as evidenced by the increased transcription activity in vivo (Fig. 3b) and in vitro (Fig. 2a). Compared with either HrpR alone or HrpS alone or when HrpR and HrpS were mixed together (denoted as HrpR+HrpS, Fig. 3c), co-purified HrpRS bound the UAS DNA with greater affinity (Fig. 3d), indicating co-operative binding to the PhrpL. Lack of increased UAS binding in the presence of pre-mixed, separately purified HrpR and HrpS, probably reflects incompetent subunit assembly (in line with the in vitro transcription assays; Fig. 2b). These features of the hrpL promoter are in good agreement with other EBP interactions at σ54-dependent promoters in terms of co-operative binding to the UAS and exclude promoter architecture as the major basis of HrpRS co-dependency43.

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