activator of one protease transforms into inhibitor of...

Activator of one protease transforms into inhibitor of another in

response to nutritional signals

Jinki Yeom1 and Eduardo A. Groisman1,2

1 Department of Microbial Pathogenesis, Yale School of Medicine, 295 Congress Avenue,

New Haven, CT 06536, USA

2 Yale Microbial Sciences Institute, P.O. Box 27389, West Haven, CT, 06516, USA

Corresponding author: [email protected]

Supplementary Material

Supplemental figures S1–S8

Supplemental tables S1–S3

2

Supplemental Figure S1. The protease adaptor ClpS binds and stabilizes the HspQ

protein, inhibiting proteolysis by ClpSAP. (A) Stability of the ClpS-HA protein in clpS-

HA (JY691) and clpS-HA hspQ (JY699) Salmonella. Protein synthesis was inhibited with

tetracycline (50 µg/ml). Samples were removed at the indicated times and analysed by

Western blotting with antibodies directed to the HA tag of the OmpA protein. (B)

Stability of the HspQ-FLAG protein in hspQ-FLAG (JY674) and hspQ-FLAG clpS (JY696)

Salmonella. Protein synthesis was inhibited and analysed as in (A) except that

antibodies directed to the AtpB protein were used in place of those directed to the

OmpA protein. (C) Interaction between HspQ-FLAG, with either ClpS-HA or RssB-HA

synthesized using an in vitro transcription/translation system. Proteins were incubated

at room temperature for 2 h, and then with anti-FLAG and anti-HA magnetic beads for

A

ClpS-HA

hspQ

After Tc (min)

OmpA

0 15 30 60 120 0 15 30 60 120

HspQ-FLAG

clpS0 15 30 60 120 After Tc (min)

AtpB

0 15 30 60 120

B

ClpS-HAHspQ-FLAG

Input Pull-down withanti-HA

Pull-down withanti-FLAG

HspQ-FLAG

RssB-HA

Detected withanti-FLAG

49(kDa)

28

14

RssB-HA+ - -- + -

--

+-

-+

- - +- + ++ - -- + -

--

+-

-+

- - +- + ++ - -- + -

--

+-

-+

- - +- + +

ClpS-HA

Detected withanti-HA

49

28

14

PhoP

0 1 2 4

ClpAP

Time (h)

ClpAPK

ClpP

HspQClpS

ClpSAPClpSAPHspQ

0 1 2 4 0 1 2 4

D E

Yeom_FigS1

C

T25-

Hsp

Q

T18c

T18c-ClpST18c-RssB

T18c-ClpAT18c-ClpP

T25-Zip/T18c-Zip

3

an additional 2 h. Immunoprecipitated samples were analysed using anti-FLAG and

anti-HA antibodies. Data are representative of two independent experiments, which

gave similar results. Boxes highlight relevant bands. (D) Spots of E. coli strain BTH101

harboring plasmid pKT25-HspQ with either pUT18c-RssB, pUT18c-ClpS, pUT18c-ClpA

or pUT18c-ClpP. Negative control strains carry the plasmid vector along with pKT25-

HspQ. Positive control strains carry pKT25-Zip and pUT18c-Zip. (E) SDS-PAGE

analysis for time course in vitro degradation of the PhoP protein (0.5 µM) mixed with

ClpA (0.08 µM), ClpP (0.2 µM) in the absence or presence of ClpS (1.0 µM) and HspQ

(0.5 µM). All reactions were carried out at 30˚C for the indicated times in the presence

of an ATP regeneration system and started by the addition of substrates. After

incubation, protein amounts were determined by Coomassie-staining following

separation on a 4-12% SDS-PAGE gel. Data are representative of two independent

experiments, which gave similar results. See also Fig. 2.

4

Supplemental Figure S2. The Salmonella HspQ protein is a Lon substrate and Lon-

enhancing factor. (A) SDS-PAGE analysis for time course in vitro degradation of the

HspQ and Hha proteins. His-Hha (0.5 µM) was mixed with Lon (0.2 µM) in the absence

or presence of HspQ (0.5 µM). Reactions were carried out at 30˚C for the indicated

times in the presence of an ATP regeneration system and started by the addition of

substrates. After incubation, protein amounts were determined by Coomassie-staining

following separation on a 4-12% SDS-PAGE gel. Data are representative of three

independent experiments, which gave similar results. (B) Degradation of the His-Hha

protein in (A) was determined by quantification of bands. Relative His-Hha levels were

calculated from three independent experiments. (C) Western blot analysis of crude

extracts from hspQ-FLAG (JY674), hspQ-FLAG clpS (JY696), hspQ-FLAG clpA (JY741),

hspQ-FLAG clpX (JY701) and hspQ-FLAG lon (JY703) Salmonella. Samples were analysed

using antibodies directed to the FLAG tag of the AtpB protein. Data are representative

HspQ

0 1 2 4 Time (h)

Lon

PK

His-Hha

LonHha

0 1 2 4 0 1 2 4 0 1 2 4

LonHspQ

LonHspQ+HhaHha

A

HspQ-FLAG

AtpB

C

Yeom_FigS2

B

0 1 2 3 40

20406080

100120

Time (h)

His-

Hha

rem

aing

pro

tein

s (%

of i

nitia

l)

Hha

Hha + Lon

Hha + HspQ + Lon

1.0 0.4 0.8 1.0 4.4

PhoP

Time (h)

LonPK

HspQ

LonLon

HspQ0 1 2 4 0 1 2 4

D

5

of three independent experiments, which gave similar results. (D) SDS-PAGE analysis

for time course in vitro degradation of the PhoP protein. PhoP (0.5 µM) was mixed with

Lon (0.2 µM) in the absence or presence of HspQ (0.3 µM). Reactions were carried out

at 30˚C for the indicated times in the presence of an ATP regeneration system and

started by the addition of substrates. After incubation, protein amounts were

determined by Coomassie-staining following separation on a 4-12% SDS-PAGE gel.

Data are representative of two independent experiments, which gave similar results.

See also Fig. 2.

6

Supplemental Figure S3. Identification of the HspQ acetylation site, predicted

structures of the Salmonella HspQ and Qad proteins, and genetic organization of the

hspQ and qad genes. (A) High-resolution tandem mass spectrometry analysis of the

HspQ protein revealed acetylation at Lys96 (red arrow). (B) Structural model of the

Salmonella HspQ protein based on the structure of the Escherichia coli HspQ protein

(PDB: 5ycq). Acetylated Lys96 is shown in red. (C) Diagram of the qad (STM14_1223)-

hspQ chromosomal region. (D) The Salmonella qad gene specifies a 136 amino acid long

protein predicted to be a CoA binding domain (NCBI: COG1832). (E) Structural model

of the Salmonella Qad protein based on the structure of the Klebsiella Qad protein (PDB:

2duw). See also Fig. 3.

11/21/16, 5:11 PM

Page 1 of 1file:///Users/jinkiyeom/Desktop/__data_20161116_F498267.dat_q936_p1_mz=10.07-830.83_scoring.svg

AC

K Q L

b2

Q

b3

A

b4

P

y1

b5y(

1)

b(3)

b(2)

b*(4

)b(

4)

b(5)

200 400 600 800

m/z

0

20

40

60

80

100

% o

f bas

e pe

ak

0.0e+0

5.0e+3

1.0e+4

1.5e+4

2.0e+4

2.5e+4

3.0e+4

ion current

Lys96

D E

CoA

B

C

Qad

1 50 100 136

CoA binding domain

A

qad hspQ

Yeom_FigS3

7

Supplemental Figure S4. Acetylation of HspQ is critical for Lon-mediated

degradation. (A-C) Degradation of the HspQ (A), His-Hha (B) and PhoP (C) proteins in

Fig. 3F was determined by quantification of bands. Relative protein levels were

calculated from two independent experiments. (D) SDS-PAGE analysis for time course

in vitro degradation of PhoP (0.5 µM) and Hha (0.3 µM) in absence of acetyl-CoA.

Substrates were mixed with ClpA (0.08 µM), ClpP (0.2 µM), ClpS (0.5 µM) and/or Lon

(0.2 µM). HspQ acetylation was achieved by preincubating HspQ (0.5 µM) with Qad

(0.5 µM) and Pat (0.2 µM) at 37˚C for 3 h. Reactions were carried out at 30˚C for the

indicated times in the presence of an ATP regeneration system and started by the

addition of substrates. After incubation, protein amounts were determined by

Coomassie-staining following separation on a 4-12% SDS-PAGE gel. Data are

A

C

0 1 2 3 40

20406080

100120

Time (h)

HspQ

rem

aing

pro

tein

s (%

of i

nitia

l)HspQ + LonHha + HspQ + LonHspQAc + LonHha + HspQAc + Lon

0 1 2 3 40

20406080

100120

Time (h)

Hha

rem

aing

pro

tein

s (%

of i

nitia

l)

Hha + HspQ + Lon

Hha + HspQAc + Lon

0 1 2 3 40

20406080

100120

Time (h)

PhoP

rem

aing

pro

tein

s (%

of i

nitia

l) ClpAPSHspQ + ClpAPSLon + HspQ + ClpAPSHha + Lon + HspQ + ClpAPSHha + Lon + HspQAC + ClpAPS

B

DYeom_FigS4

0 1 2 4

HspQ

Hha

Time (h)

PK

ClpAPSHspQLon

Lon

ClpAPSHspQ

Hha

Lon

ClpS

ClpPPhoP

ClpA

0 1 2 4 0 1 2 4 0 1 2 4 0 1 2 4 0 1 2 4Pat

Qad

ClpAPSHspQLon

HspQLon

HhaHspQLon

Lon

8

representative of two independent experiments, which gave similar results. See also

Fig. 3.

9

Supplemental Figure S5. HspQ acetylation status impacts proteolysis by Lon but not

by ClpSAP. (A) SDS-PAGE analysis of in vitro degradation of His-Hha (0.5 µM) by Lon

(0.2 µM) in the absence or presence of the HspQ (0.5 µM), HspQR (0.5 µM) or HspQQ

(0.5 µM) proteins. Proteins were visualized by Coomassie-staining following separation

on a 4-12% SDS-PAGE gel. Data are representative of three independent experiments,

which gave similar results. (B) Degradation of the HspQ proteins in (A) was

determined by quantification of bands. Relative HspQ levels were calculated from

three independent experiments. (C) Interactions among the ClpS-His, HspQ-FLAG,

HspQR-FLAG and HspQQ-FLAG proteins synthesized using an in vitro

transcription/translation system. Proteins were incubated with purified ClpS-His at

room temperature for 2 h, and then with anti-FLAG and anti-His magnetic beads for an

A B

PhoP

ClpAP0 1 2 4 Time (h)

ClpAPK

ClpP

HspQClpS

ClpSAPClpSAPHspQ

ClpSAPHspQR

ClpSAPHspQQ

0 1 2 4 0 1 2 4 0 1 2 4 0 1 2 4ClpS-HisHspQ-FLAG

Input Pull-down withanti-His

Pull-down withanti-FLAG

HspQ-FLAG

Detected withanti-FLAG

49(kDa)

28

14

Detected withanti-His

4928

14 ClpS-His

-- ++-

- + ++ R Q-

-+

R Q-- +

+-- + +

+ R Q-

-+

R Q-- +

+-- + +

+ R Q-

-+

R Q

HspQ

0 1 2 4 Time (h)

Lon

PK

His-Hha

HspQHha

HspQ HspQRHha

HspQR HspQQHha

HspQQ0 1 2 4 0 1 2 4 0 1 2 4 0 1 2 4 0 1 2 4

C

Yeom_FigS5

D

0 1 2 3 40

20406080

100120

Time (h)

HspQ

rem

aing

pro

tein

s (%

of i

nitia

l)

HspQ

HspQ + Hha

HspQR

HspQR + Hha

HspQQ

HspQQ + Hha

10

additional 2 h. Immunoprecipitated samples were analysed using anti-FLAG and anti-

His antibodies. Data are representative of two independent experiments, which gave

similar results. (D) SDS-PAGE analysis of time course in vitro incubation of the PhoP

protein (0.5 µM) with ClpA (0.08 µM) and ClpP (0.2 µM) in the absence of ClpS, or with

ClpS (1.0 µM) and no HspQ, HspQ (0.5 µM), HspQR (0.5 µM) or HspQQ (0.5 µM).

Substrate degradation assay was analysed as in (A). Data are representative of three

independent experiments, which gave similar results. See also Fig. 3 and 4.

11

Supplemental Figure S6. The HspQ protein does not alter the abundance of ClpS-

independent ClpAP substrates. (A) Stability of the AcnB protein in wild-type (14028s),

clpX (JY649), clpA (JY650) and clpS (JY651) Salmonella. Protein synthesis was inhibited

with tetracycline (50 µg/ml). Samples were removed at the indicated times and

analysed by Western blotting with antibodies directed to the AcnB protein. Data are

representative of three independent experiments, which gave similar results. (B)

Western blot analysis of crude extracts prepared from oat-FLAG (JY655), oat-FLAG clpS

(JY657), oat-FLAG hspQ (JY686) and oat-FLAG qad (JY774) Salmonella with a gfp-laa

expressing plasmid (pFPV25-gfp-laa). Samples were analysed using antibodies directed

to the FLAG tag, the GFP protein or the GroEL protein. Data are representative of two

independent experiments, which gave similar results. (C) Western blot analysis of

crude extracts prepared from oat-FLAG (JY655), oat-FLAG clpS (JY657) and oat-FLAG

0 1 2 4

clpX

After Tc (h)0 1 2 4 0 1 2 4 0 1 2 4

clpA clpSwild-type

Oat-FLAG

GroEL

AcnBOat-FLAG

GFP-LAA

GroEL

A

B C

AcnB

Yeom_FigS6

0 10 30 60

hspQ

After Tc (min)

clpPwild-typeD

GFP-LAA0 10 30 60 0 10 30 60

12

clpA (JY902) Salmonella. Samples were analysed using antibodies directed to the FLAG

tag, the AcnB protein or the GroEL protein. Data are representative of three

independent experiments, which gave similar results. (D) Stability of the GFP-LAA

protein in wild-type (14028s), hspQ (JY683) and clpP (JY186) Salmonella with a gfp-laa

expressing plasmid (pFPV25-gfp-laa). Protein synthesis was inhibited with tetracycline

(50 µg/ml). Samples were removed at the indicated times and analysed by Western

blotting with antibodies directed to the GFP protein. Data are representative of two

independent experiments, which gave similar results. See also Fig. 5.

13

Supplemental Figure S7. mRNA abundances of the oat and hha genes are similar in

wild-type, hspQ, and qad Salmonella when grown on glucose or glycerol. (A) mRNA

abundance of the oat gene produced by oat-FLAG (JY655), oat-FLAG hspQ (JY686), and

oat-FLAG qad (JY774) Salmonella grown on glycerol or glucose. (B) mRNA abundance of

the hha gene produced by oat-FLAG (JY655), oat-FLAG hspQ (JY686), and oat-FLAG qad

(JY774) Salmonella with a His-hha-expressing plasmid (pHis-hha) following growth on

glycerol or glucose. his-hha transcription from pHis-hha was induced with IPTG (100

µM). For all qRT-PCR analysis, mRNA abundance was normalized to those of the ompA

gene. The sequence of the primers used in qRT-PCR are presented in Table S3. Data

shown are the mean and SD from three independent experiments. See also Fig. 6.

wild-ty

pehspQ qa

d

wild-ty

pehspQ qa

d0.0

0.2

0.4

0.6

0.8

oat

mRN

A ab

unda

nce

glycerol glucose

wild-ty

pehspQ qa

d

wild-ty

pehspQ qa

d0

1

2

3

hha

mRN

A ab

unda

nce

glycerol glucose

A B

Yeom_FigS7

14

Supplemental Figure S8. The deduced amino acid sequence of the qad gene is

conserved among members of the family Enterobacteriaceae. Alignment of the deduced

amino acid sequences of the hspQ genes from the listed species of the family

Enterobacteriaceae. Red arrow indicates Lys96, which is acetylated in the Salmonella

HspQ protein. Y. pestis harbors a His at position 96.

CLUSTAL 2.1 MULTIPLE SEQUENCE ALIGNMENTFile: /Users/younsoosim/Desktop/hspQ/STM.ps Date: Fri Oct 5 13:49:27 2018 Page 1 of 1

**:**:********.* *:***::*:** *** * *::* * **: ******:*.::* .:********* * : *****::**** :**:*****:***ACY87717.1 MIASKFGIGQQVRHSLLGYLGVVVDIDPEYSLDEPSPDELAVNDELRAAPWYHVVMEDDDGQPVHTYLAEAQLRSEMRDEHPEQPSMDELARTIRKQLQAPRLRN 105CCC29975.1 MIASKFGIGQQVRHSLLGYLGVVVDIDPEYSLDEPSPDELAVNDELRAAPWYHVVMEDDDGQPVHTYLAEAQLRSEMRDEHPEQPSMDELARTIRKQLQAPRLRN 105AIZ91912.1 MIASKFGIGQQVRHSLLGYLGVVVDIDPVYSLSEPSPDELAVNDELRAAPWYHVVMEDDNGLPVHTYLAEAQLSSELQDEHPEQPSMDELAQTIRKQLQAPRLRN 105CDX06253.1 MIASKFGIGQQVRHSLLGYLGVVVDIDPVYSLSEPSPDELAVNDELRAAPWYHVVMEDDNGLPVHTYLAEAQLSSELQDEHPEQPSMDELAQTIRKQLQAPRLRN 105CBG87807.1 MIASKFGIGQQVRHSLLGYLGVVMDIDPVYSLEEPSPDELAVNDELRAAPWYHVVMEDDDGQPVHTYLAEAQLRSETQDEHPEQPSMDELAQTIRKQLQAPRLRN 105KML25937.1 MIASKFGIGQQVRHTLLGYLGVVVDIDPEYSLDEPSADELAVNAELRAAPWYHVVMEGDDGQPVHTYLAEAQLSSELQEEHPEQPTMDELAQTIRKQLQAPRLRN 105AVH16492.1 MIASKFGIGQQVRHTLLGYLGVVVDIDPEYSLDEPSADELAVNAELRAAPWYHVVMEGDDGQPVHTYLAEAQLSSELQEEHPEQPTMDELAQTIRKQLQAPRLRN 105ADF62221.1 MIASKFGIGQQVRHTLLGYLGVVVDIDPEYSLDEPSADELAVNAELRAAPWYHVVMEGDDGQPVHTYLAEAQLSSELQDEHPEQPTMDELAQTIRKQLQAPRLRN 105AHJ74447.1 MIASKFGIGQQVRHSLLGYLGVVVDIDPEYSLDEPSADELAVNDELRALPWYHVVMEDDDGQPVHTYLAEAQLTSEITDEHPEQPSMDELARTIRRQLQAPRLRN 105WP_062777489.1 MITSKFGIGQQVRHSLLGYLGVVVDIDPVYSLDEPEPDDLAANDELRALPWYHVVMEDDEGQPMHTYLAEAQLSSEPRDDHPEQPTMDELARTIRRQLQAPRLRN 105AIR03971.1 MIASKYGIGQQVRHSLLGYLGVVVDIDPEYSLDEPQEDDLADNSALRAAPWYHVVMEDDDGQAIHTYLAEAQLSSEDDDEHPEQPSMDELAASIRQQLQAPRLRN 105ALB51068.1 MIASKFGIGQQVRHSLLGYLGVVVDIDPEYSLDEPEVDELAVNAELRAAPWYHVVMEDDDGQPVHTYLAEAQLSGEMQEEHPEQPSMDELARSIRQQLQAPRLRN 105AJF72496.1 MIASKFGIGQQVRHTLLGFLGVVVDIDPEYSLAEPAEDEIAANDELRALPWYHVVMEDEDGQPVHTYLAEAQLSSEPSDEHPEQPSMDELARTIRQQLQAPRLRN 105APS95917.1 MIASKFGIGQQVRHTLLGYLGVIVDVDPEYSLAEPEEDEIAANDELRAAPWYHVVMEDDDGQPIHTYLAEAQLSSETRDEHPEQPSLDELAKTIRQQLQAPRLRN 105AVR06007.1 MIASKFGIGQQVRHSLLGYLGVIVDIDPEYSLGAPDADEIAGNDALRAAPWYHVVMEDDDGQPVHTYLAEAQLRGEAHDEHPEQPSMDELAQTIRRQLQAPRLRN 105ASL97103.1 MIASKFGIGQQVRHKLLGYLGVVIDIDPEYSLEQPKADEIAANDELRSAPWYHVVMEDEEGQPVHTYLAEAQLDSEAQEAHPEQPSLDELAESIRHQLQAPRLRN 105[Yersinia.fasta] MIASKFGIGQQVRHSLHGYLGVVIDIDPEYSLAPPEPDEVANNKTLRSSPWYHVVIEDDDGQPVHTYLAEAQLTYEDVDAHPEQPSLDELAASIRHQLQAPHLRN 105 1.......10........20........30........40........50........60........70........80........90.......100.....

Lelliottia nimipressuralisLeclercia adecarboxylata

Raoultella ornithinolytica

Salmonella entericaSalmonella bongori

Escherichia coliShigella flexneri

Citrobacter rodentium

Kluyvera intermediaKosakonia sacchari

Enterobacter cloacae

Cronobacter sakazakiiCedecea neteri

Klebsiella pneumoniae Pluralibacter gergoviae

Serratia marcescensYersinia pestis

Yeom_FigS8

15

Supplemental Table S1. Genome analysis of Enterobacteriaceae species with

conserved asynteny between the hspQ and qad genes.

1 tBLASTn analysis for conserved qad and hspQ in bacterial genomes.

Enteric bacteria species in which qad and hspQ are conserved1 Lys96 residue in HspQ

Salmonella enterica O

Salmonella bongori O

Escherichia coli O

Citrobacter rodentium O

Shigella flexneri O

Enterobacter cloacae O

Cronobacter sakazakii X

Klebsiella pneumoniae X

Serratia marcescens X

Yersinia pestis X

Kosakonia sacchari X

Kluyvera intermedia X

Lelliottia nimipressuralis O

Leclercia adecarboxylata O

Raoultella ornithinolytica X

Pluralibacter gergoviae X

Cedecea neteri X

16

Supplemental Table S2. Bacterial strains and plasmids used in this study.

Strains Relevant characteristics Source

Escherichia coli

BL21 (DE3) F- ompT hsdS gal [lon] [dcm] (Its857 ind1 Sam7 nin5 lacUV5-T7 gene 1) (Studier and Moffatt, 1986)

DH5a Host strain used for generation and propagation of plasmid constructs Life Technologies

Salmonella enterica serovar Typhimurium

14028s wild-type (Fields et al., 1989)

EG13917 phoP-HA::cm (Shin and Groisman, 2005)

EG16039 lon::cm This study

JY186 clpP::cm This study

JY649 DclpX (Yeom et al., 2017)

JY650 DclpA (Yeom et al., 2017)

JY651 DclpS (Yeom et al., 2017)

JY655 oat-FLAG (Yeom et al., 2017)

JY657 oat-FLAG / clpS (Yeom et al., 2017)

JY674 hspQ-FLAG This study

JY683 hspQ::cm This study

17

JY686 oat-FLAG / hspQ This study

JY687 oat-FLAG / clpS / hspQ This study

JY691 clpS-HA (Yeom et al, 2018)

JY692 hspQ-FLAG / clpS-HA This study

JY694 clpA-HA (Yeom et al, 2018)

JY695 hspQ-FLAG / clpA-HA This study

JY696 hspQ-FLAG / clpS This study

JY699 clpS-HA / hspQ This study

JY700 clpA-HA / hspQ This study

JY701 hspQ-FLAG / clpX This study

JY703 hspQ-FLAG / lon This study

JY705 oat-FLAG / phoP-HA / qad This study

JY740 hspQ-FLAG / qad This study

JY741 hspQ-FLAG / clpA This study

JY754 hspQ-FLAG / lon / qad This study

JY772 oat-FLAG / clpS / hspQ / qad This study

JY773 oat-FLAG / hspQ / qad This study

18

JY774 oat-FLAG / qad This study

JY775 oat-FLAG / qad / clpS This study

JY865 oat-FLAG / hspQ-FLAG This study

JY872 oat-FLAG / phoP-HA This study

JY873 oat-FLAG / phoP-HA / hspQ This study

JY874 oat-FLAG / phoP-HA / clpS This study

JY875 oat-FLAG / phoP-HA / hspQ / clpS This study

JY889 qad::km This study

JY892 hspQK96R-FLAG This study

JY893 hspQK96R-FLAG / qad This study

JY894 hspQK96Q-FLAG This study

JY895 hspQK96Q-FLAG / qad This study

JY898 hspQ-FLAG / pat This study

JY899 hspQ-FLAG / pat / qad This study

JY902 oat-FLAG / clpA This study

JY1014 oat-FLAG / lon This study

JY1015 oat-FLAG / lon / clpS This study

19

JY1016 oat-FLAG / lon / hspQ This study

JY1017 oat-FLAG / qad / lon This study

JY1018 oat-FLAG / lon / hspQ / qad This study

JY1019 hspQ-FLAG / lon / clpS This study

JY2000 hspQK96R-FLAG /lon This study

JY2001 hspQK96Q-FLAG /lon This study

JY2002 oat-FLAG / hspQK96R-FLAG This study

JY2003 oat-FLAG / hspQK96Q-FLAG This study

Plasmids

pKD46 reppSC101ts AmpR ParaBAD-gbexo (Datsenko and Wanner,

2000)

pKD3 repR6Kg AmpR FRT CmR FRT (Datsenko and Wanner, 2000)

pKD4 repR6Kg AmpR FRT KmR FRT (Datsenko and Wanner, 2000)

pCP20 reppSC101ts l cI857 FLP AmpR CmR (Datsenko and Wanner,

2000)

pUHE-21-2-lacIq reppMB1 lacIq AmpR (Soncini et al., 1996)

pUHE-hspQ reppMB1 lacIq AmpR Plac-hspQ This study

pUHE-qad reppMB1 lacIq AmpR Plac-qad This study

pUHE-His-lon reppMB1 lacIq AmpR Plac-his-lon This study

20

pUHE-His-hha reppMB1 lacIq AmpR Plac-his-hha This study

pUHE-ftsA-FLAG reppMB1 lacIq AmpR Plac-ftsA-FLAG (Yeom et al, 2018)

pFPV25-gfp pBR332 AmpR p-gfpmut3 (Valdivia and Falkow,

1996)

pFPV25-gfp-laa pBR332 AmpR p-gfpmut3-laa This study

pET28+a pBR332 lacI KmR pT7 Novagen

pET28+a-clpS pBR332 lacI KmR pT7- clpS (Yeom et al, 2018)

pET28+a-clpA pBR332 lacI KmR pT7- clpA (Yeom et al, 2018)

pET28+a-clpP pBR332 lacI KmR pT7- clpP (Yeom et al, 2018)

pET28+a-hspQ pBR332 lacI KmR pT7- hspQ This study

pET28+a-qad pBR332 lacI KmR pT7- qad This study

pET28+a-pat pBR332 lacI KmR pT7- pat This study

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Supplemental Table S3. Primers used for strain or plasmid constructions or qRT-PCR

or to generate templates for in vitro transcription/translation.

No. Purpose Sequence (from 5' to 3')

1550 Amplification of gfp-laa gene for unstable GFP expression CACCTGACGTCTAAGAAACC

3071 Amplification of gfp-laa gene for unstable GFP expression

CATTAAAGCTTGCATGCCTGCAGGAGATTTAAGCTGCTAAAGCGTAGTTTTCGTCGTTTGCTGCAGGCCTTTTGTATAGTTCATCCATGC

3815 lon inactivation in Salmonella ATCTGATTACCTGGCGGACACTAAACTAAGAGAGAGCTCTTGTAGGCTGGAGCTGCTTCG

3816 lon inactivation in Salmonella TGCCAGCCCTGTTTTTATTAGCGCTATTTGCGCGAGGTCACATATGAATATCCTCCTTAG

15054 qRT-PCR of ompA gene in Salmonella GGGCTGGTCTCAGTACCATGA

15055 qRT-PCR of ompA gene in Salmonella TCATGAGTCGGGCCATCA

15949 In vitro synthesis of ClpS-HA from Salmonella

GCGAATTAATACGACTCACTATAGGGCTTAAGTATAAGGAGGAAAAAATATGGGTAAGACGAACGATTGGCTGGATTTTGACCAGTTGGTGGAA

15966 qRT-PCR of oat gene in Salmonella GGCCCTCTGGAGCTCATTTT

15967 qRT-PCR of oat gene in Salmonella GGTTTAGCGTTCGCTTCTCG

16044 Insertion FLAG tag at C-terminus of hspQ gene in

Salmonella

CAAGCAGCTTCAGGCGCCGCGACTACGTAACGACTACAAGGACGACGATGACAAGTGAGTGTAGGCTGGAGCTGCTTC

16045 Insertion FLAG tag at C-terminus of hspQ gene in

Salmonella

GTTTAAATGGCCGGATATCGCTTTCCGGCCTTTTAAATACATATGAATATCCTCCTTAGT

16046 hspQ inactivation in Salmonella

TATAAAGGGTATCTATTTCCCGGGAGGTGACTGTGTAGGCTGGAGCTGCTTC

16047 hspQ inactivation in Salmonella

AATGGCCGGATATCGCTTTCCGGCCTTTTAAATACAATATGAATATCCTCCTTAGT

22

16052 Amplification of hspQ gene

from Salmonella for expression

GCGGGATCCAATGATAGCCAGCAAATTCGGTATCG

16053 Amplification of hspQ gene


GCGAAGCTTTCAGTTACGTAGTCGCGGCGCCTGAAGCTG

16284 Amplification of qad gene

from Salmonella for protein purification

CGCGGATCCATGATGAAAGAGACCGATATTGCTGATGTTTTGACG



CTCCTCGAGTTTTCGCCAGCCCCAGGCGAGGGATCTCAATCGCCG



GCGGGATCCAATGATGAAAGAGACCGATATTGCTGATGTTTTGA



GCGAAGCTTTTATTTCGCCAGCCCCAGGCGAGGGATCTCAATCG

16313 Amplification of pat gene


CGCGGATCCATGAGCCAGCAAGGACTGGAAGCGCTACTGCGACC

16314 Amplification of pat gene


CGCCTCGAGTCACGATTCATCACATTTGGCCAGATTCAGCGT

16629 Amplification of his-hha gene


GCGGGATCCAATGCATCATCACCATCACCACTCTGATAAACCATTAACTAAAACTGATTA

16630 Amplification of his-hha gene


GCGAAGCTTTTAACGAATGAATTTCCATACTGAAGAGGG

16924 qad inactivation in Salmonella TTTGTCGTACACTTTGCAAAACAGCCAGGAGAAAG GTGTAGGCTGGAGCTGCTTC

16925 qad inactivation in Salmonella TTAAAAGGCCGGAAAGCGATATCCGGCCATTTAAACTT TATGAATATCCTCCTTAGT

16926 pat inactivation in Salmonella GTTTAAAATTATCCGGTCACTTCTGTGTAAGGGAAACCGGT GTGTAGGCTGGAGCTGCTTC

16927 pat inactivation in Salmonella TCAGTACCCGTTAAAGTGGTCAACATTTCCAGTACATTAC TATGAATATCCTCCTTAGT

16928 Substitution of lysine with arginine at position 96 in

Salmonella HspQ

TGAACTGGCGCGTACCATTCGCCGTCAGCTTCAGGCGCCGCGACTACGTAACGACTACAAGGACGACGATGACAAGTGA GTGTAGGCTGGAGCTGCTTC

23

16929 Substitution of lysine with glutamine at position 96 in

Salmonella HspQ

TGAACTGGCGCGTACCATTCGCCAGCAGCTTCAGGCGCCGCGACTACGTAACGACTACAAGGACGACGATGACAAGTGA GTGTAGGCTGGAGCTGCTTC

16934 Amplification of hspQ gene from Salmonella for protein

purification GCGCATATGATGATAGCCAGCAAATTCGGTATCGGCCAACAGGTCCGCC

16935 Amplification of hspQ gene from Salmonella for protein

purification GCGAAGCTTTCAGTTACGTAGTCGCGGCGCCTGAAGCTGCTTGCGAATG

16936 In vitro synthesis of HspQ-FLAG from Salmonella

GCGAATTAATACGACTCACTATAGGGCTTAAGTATAAGGAGGAAAAAATATGATAGCCAGCAAATTCGGTATCGGCCAACAGGT

16937 In vitro synthesis of HspQ-FLAG from Salmonella

AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTCACTTGTCATCGTCGTCCTTGTAGTCGTTACGTAGTCGCGGCGCCTGAAGCTGCTTGCGAA

16938 In vitro synthesis of ClpS-HA from Salmonella

AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTCAAGCGTAATCTGGAACATCGTATGGGTAGGCTTTTTCCAGCGTACACAGCAACGG

16939 In vitro synthesis of RssB-HA from Salmonella

GCGAATTAATACGACTCACTATAGGGCTTAAGTATAAGGAGGAAAAAATATGACGCAGCCATTGGTCGGAAAACAGATTCTTATTGTTG

16940 In vitro synthesis of RssB-HA from Salmonella

AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTCAAGCGTAATCTGGAACATCGTATGGGTATTCCGCAGACAACATCAATCGCAGACGCCCTCCTGCGCCC

17202 qRT-PCR of hha gene in Salmonella TACAGCCAGCTCATTGTCGG

17203 qRT-PCR of hha gene in Salmonella TTTGATGCGTTTACGGCGCT

20000 clpP inactivation in Salmonella

ATGTCATACAGCGGAGAACGAGATAATTTGGCCCCTCATAGTGTAGGCTGGAGCTGCTTC

20001 clpP inactivation in Salmonella

TCAATTACGATGGGTCAAAATTGAGTCAACCAAACCGTACTATGAATATCCTCCTTAGT

activator of one protease transforms into inhibitor of...

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