Hrp regulatory proteins, HrpR, HrpS, HrpV

1
Hrp regulatory proteins, HrpR, HrpS, HrpV of
Pseudomonas syringae
2
Ph.D., 2004, P.C. Burrows
3
activation of Es54-dependent transcription
M. Buck et al., 2006, Biochem. Soc. Transactions
4
domain organisation of AAA s54 activators
C1
C7
C6
C5
C4
C3
C2
HTH
NH3
COOH
DNA binding domain
Central AAA domain
Regulatory domain
domain organisation of AAA s54 activators
lacking N-terminal domain
DNA binding domain
AAA domain
PspF
C1
C7
C6
C5
C4
C3
C2
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
R
S
R
S
hrpL
other genes regulated by HrpR and HrpS
s54
PL
Type III Protein Export Complex
hrp regulon
6
chromosomal single copy reporter system in E.
coli
-
V
S
R
1
R
S
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
C7
C6
C5
C4
C3
C2
HTH
NH3
COOH
DNA binding domain
Central AAA domain
Regulatory domain
domain organisation of AAA s54 activators
lacking N-terminal domain
DNA binding domain
AAA domain
PspF
C1
C7
C6
C5
C4
C3
C2
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
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
C7
C6
C5
C4
C3
C2
NH3
COOH
HTH
Walker A
GAFTGA
Walker B
R finger
Sensor II
nucleotide binding
s54 binding
nucleotide hydrolysis
intersubunit catalysis
nucleotide dependent oligomerisation
86
42
107 108
162
168
227
PspF
GERGTGKEL
GAFTGA
DELAT
FRADLLDRL
WPGNIRELKNVVE
45
170
228
88
109 110
164
228
228
228
HrpR
GETGTGKDT
GAFTGV
DEIDS
FRRDLFFRL
WPGNIRELKSAAK
LPRRTLYHRMKEL
43
226
168
162
86
107 108
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
C7
C6
C5
C4
C3
C2
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
88
109 110
170
164
HrpR
GETGTGKDT
GAFTGV
DEIDS
FRRDLFFRL
WPGNIRELKSAAK
LPRRTLYHRMKEL
hrpL-lacZ activity in the presence of WT HrpS

-
-
-
-
-
-
6
6
226
43
168
162
86
107 108
HrpS
GETGTGKDT
GAYTGA
DEIDS
FRRDLYFRL
WPGNIRELKAAAK
D HTH
-
-
-
hrpL-lacZ activity in the presence of WT HrpR
8
16

14
34
red letters - mutations which abolish hrpL
transcription blue letters mutations which
diminish hrpL 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
-600
-24
-12
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 0.1SDS 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
14
(No Transcript)
15
(No Transcript)
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
s54
S
lacZ
hrpL-lacZ chromosomal fusion
S
R
S
R
?
?
s54
R
S
s54
R
S
lacZ
lacZ
17
Domain organisation of AAA s54 activators
C1
C7
C6
C5
C4
C3
C2
HTH
NH3
COOH
DNA binding domain
Central AAA domain
Regulatory domain
Domain organisation of AAA s54 activators
lacking N-terminal domain
AAA domain
DNA binding domain
PspF
C1
C7
C6
C5
C4
C3
C2
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
88
164
170
228
109 110
HrpR
GETGTGKDT
GAFTGV
DEIDS
FRRDLFFRL
WPGNIRELKSAAK
226
168
162
43
86
107 108
HrpS
GETGTGKDT
GAYTGA
DEIDS
FRRDLYFRL
WPGNIRELKAAAK
C1-C7 seven conserved sequences of s54
activators