Hrp regulatory proteins, HrpR, HrpS, HrpV of Pseudomonas syringae

1. Hrp regulatory proteins, HrpR, HrpS, HrpV of Pseudomonas syringae

2. A) s70 Dependent Transcription Initiation Activator required for promoter engagement -35-10 -35 -10 Closed Complex Open Complex Spontaneous B) s54 Dependent Transcription Initiation Activator driven nucleotide hydrolysis to allow RNAP isomerisation -24-12+1 -24 -12+1 ClosedComplex OpenComplex Ph.D., 2004, P.C. Burrows

3. -24 -12 +1 Enhancer binding protein AAA+ Activator DNA Looping Event Core RNAP ATP Hydrolysis s54 +ATP IHF Enhancer DNA ~ -150 -24 -12 +1 -24 -12 +1 Closed Complex Open Complex activation of Es54-dependent transcription M. Buck et al., 2006, Biochem. Soc. Transactions

4. domain organisation of AAA+s54 activators C1 C2 C3 C4 C5 C6 C7 HTH COOH NH3 DNA binding domain Regulatory domain Central AAA+ domain domain organisation of AAA+s54 activators lacking N-terminal domain DNA binding domain AAA+ domain PspF C1 C2 C3 C4 C5 C6 C7 HrpR COOH NH3 HrpS HTH Walker A GAFTGA Walker B R finger Sensor II nucleotide binding s54 binding nucleotide hydrolysis intersubunit catalysis nucleotide dependent oligomerisation C1-C7 – seven conserved sequences of s54 activators

5. The Pseudomonas syringae Hrp regulatory system hrpR hrpS s70 PRS _ V R S R S hrpL other genes regulated by HrpR and HrpS s54 PL sL Type III Protein Export Complex hrp regulon

6. chromosomal single copy reporter system in E. coli - V S R +1 S R s54 lacZ hrpL-lacZ chromosomal fusion • - s54 dependent • requires HrpR and HrpS proteins for activity • negatively regulated by HrpV in the presence of HrpR and HrpS proteins

7. activation and negative regulation of hrpL promoter MC4100 f(hrpL-lacZ) 0.5mM IPTG 0.4% ara

8. domain organisation of AAA+s54 activators C1 C2 C3 C4 C5 C6 C7 HTH COOH NH3 DNA binding domain Regulatory domain Central AAA+ domain domain organisation of AAA+s54 activators lacking N-terminal domain DNA binding domain AAA+ domain PspF C1 C2 C3 C4 C5 C6 C7 HrpR COOH NH3 HrpS HTH Walker A GAFTGA Walker B R finger Sensor II nucleotide binding s54 binding nucleotide hydrolysis intersubunit catalysis nucleotide dependent oligomerisation C1-C7 – seven conserved sequences of s54 activators

9. temperature dependent activity of hrpL-lacZ fusion MC4100 f(hrpL-lacZ) 0.5mM IPTG s54 mutant P.s. 28oC T (oC)

10. R S S R ? ? S R R S s54 s54 lacZ lacZ do both, HrpR and HrpS have to be fully functional for co-regulation what are the effects of changing amino acids in HrpR and HrpS DNA binding domain AAA+ domain C1 C2 C3 C4 C5 C6 C7 COOH NH3 HTH Walker A GAFTGA Walker B R finger Sensor II nucleotide binding s54 binding nucleotide hydrolysis intersubunit catalysis nucleotide dependent oligomerisation 42 86 107 108 162 168 227 PspF GERGTGKEL GAFTGA DELAT FRADLLDRL WPGNIRELKNVVE 45 228 170 88 164 109 110 228 228 228 HrpR GETGTGKDT GAFTGV DEIDS FRRDLFFRL WPGNIRELKSAAK LPRRTLYHRMKEL 43 107 108 226 86 162 168 HrpS GETGTGKDT GAYTGA DEIDS FRRDLYFRL WPGNIRELKAAAK D HTH

11. functional determinants of HrpR and HrpS important for in vivo activation of the hrpL promoter in E. coli AAA+ domain DNA binding domain C1 C2 C3 C4 C5 C6 C7 COOH NH3 HTH Walker A GAFTGA Walker B R finger Sensor II nucleotide binding s54 binding nucleotide hydrolysis intersubunit catalysis nucleotide dependent oligomerisation 45 228 170 88 109 110 164 HrpR GETGTGKDT GAFTGV DEIDS FRRDLFFRL WPGNIRELKSAAK LPRRTLYHRMKEL hrpL-lacZ activity in the presence of WT HrpS - - - - - - + 6% + 6% 226 43 86 162 168 107 108 HrpS GETGTGKDT GAYTGA DEIDS FRRDLYFRL WPGNIRELKAAAK D HTH - - - hrpL-lacZ activity in the presence of WT HrpR + 8% + 14% + + 16% + 34% red letters - mutations which abolish hrpL transcription blue letters – mutations which diminishhrpL transcription amino acids are substituted with alanine

12. in vivo hrpL promoter analysis R S +1 R S s54 lacZ -24 -12 UAS hrpL-lacZ transcription fusion MU +1 lacZ -24 -12 -600 IHF 21458 -400 20930 -300 21030 -200 19200 -100 300 10

13. b-Galactosidase assay -prepare over night (O/N) culture(s) -add 0.2ml of the O/N culture(s) to the 5ml fresh media -grow cultures to mid-log phase (OD600 of 0.28-0.70) -cool the culture(s) on ice for 20 minutes -record cell density at OD600 -add aliquots of the culture(s) to the assay medium (Z buffer) -add 30ml of 0.1%SDS and 60ml of chloroform and vortex for 10 seconds -place the samples in a water bath at 28O for 5 minutes -start reaction by adding 0.2 ml of ONPG (4mg/ml) to each tube, shake for a few seconds -record the time of the reaction with a stop watch -stop reaction by adding 0.5ml of a 1M Na2CO3 -record the OD420 and OD550 for each sample OD420 – (1.75 x OD550) MU= 1000 x t x v x OD600

15. -24 -12 +1 Enhancer binding protein AAA+ Activator DNA Looping Event Core RNAP ATP Hydrolysis s54 +ATP IHF Enhancer DNA ~ -150 -24 -12 +1 -24 -12 +1 Closed Complex Open Complex

16. do both, HrpR and HrpS have to be fully functional for co-regulation what are the effects of changing amino acids in HrpR and HrpS R S + R S s54 lacZ hrpL-lacZ chromosomal fusion R S S R ? ? R R S S s54 s54 lacZ lacZ

17. Domain organisation of AAA+s54 activators C1 C2 C3 C4 C5 C6 C7 HTH COOH NH3 DNA binding domain Regulatory domain Central AAA+ domain Domain organisation of AAA+s54 activators lacking N-terminal domain AAA+ domain DNA binding domain PspF C1 C2 C3 C4 C5 C6 C7 HrpR COOH NH3 HrpS HTH Walker A GAFTGA Walker B R finger Sensor II nucleotide binding s54 binding nucleotide hydrolysis intersubunit catalysis nucleotide dependent oligomerisation 42 86 107 108 162 168 227 PspF GERGTGKEL GAFTGA DELAT FRADLLDRL WPGNIRELKNVVE 45 228 170 88 164 109 110 HrpR GETGTGKDT GAFTGV DEIDS FRRDLFFRL WPGNIRELKSAAK 43 107 108 226 86 162 168 HrpS GETGTGKDT GAYTGA DEIDS FRRDLYFRL WPGNIRELKAAAK C1-C7 – seven conserved sequences of s54 activators