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1 Supplementary Materials for Activation of master virulence regulator PhoP in acidic ph requires the Salmonella-specific protein UgtL Jeongjoon Choi and Eduardo A. Groisman* This PDF file includes: *Corresponding author. Published 29 August 2017, Sci. Signal. 10, eaan6284 (2017) DOI: /scisignal.aan6284 Fig. S1. UgtL-dependent activation of the PhoP protein in mildly acidic ph is similar in WT and hns-flag Salmonella. Fig. S2. UgtL enhances PhoP-P abundance in an MgrB-independent manner. Fig. S3. UgtL enhances PhoP-P abundance posttranscriptionally. Fig. S4. The UgtL and PhoQ proteins interact. Fig. S5. FLAG-tagged UgtL functions as WT UgtL in vivo. Fig. S6. PhoP activates transcription of the ugtl gene in mildly acidic ph and low Mg 2+ conditions. Fig. S7. UgtL promotes Salmonella resistance to magainin 2 in a PhoP-dependent manner. Table S1. Bacterial strains and plasmids used in this study. Table S2. Primers used in this study.

2 AtpB PhoP-P PhoP Fig. S1. UgtL-dependent activation of the PhoP protein in mildly acidic ph is similar in WT and hns-flag Salmonella. Phos-tag Western blot analysis of crude extracts prepared from various ugtl + and ugtl - Salmonella strains: ugtl mutant strain EG13682 (ugtl); wild type strain 14028s (WT); wild-type strain JC805, which carries hns-flag at the normal hns chromosomal location (hns-flag); and ugtl mutant strain JC925, which carries hns-flag at the normal hns chromosomal location. Blots were probed using antibodies recognizing PhoP and AtpB (loading control). Data are representative of three independent experiments, which produced similar results.

3 AtpB PhoP-P PhoP Fig. S2. UgtL enhances PhoP-P abundance in an MgrB-independent manner. Phos-tag Western blot analysis of crude extracts prepared from wild-type (WT, JC805) and ugtl (JC925), mgrb (JC969), and ugtl mgrb (JC1054) Salmonella. Blots were probed using antibodies recognizing PhoP and AtpB (loading control). Data are representative of three independent experiments, which produced similar results.

4 AtpB PhoP-P PhoP Fig. S3. UgtL enhances PhoP-P abundance posttranscriptionally. Phos-tag Western blot analysis of crude extracts prepared from Salmonella expressing phop and phoq constitutively (ugtl +, strain EG14943) and an isogenic derivative lacking the ugtl gene (ugtl -, JC1015) grown under mildly acidic conditions. Samples were analyzed using antibodies recognizing PhoP and AtpB (loading control). Data are representative of three independent experiments, which produced similar results.

5 (kda) INPUT IP αha IP αflag UgtL-HA PhoQ-FLAG PhoZ-FLAG EnvZ-FLAG αha blot UgtL-HA αflag blot PhoZ-FLAG PhoQ-FLAG EnvZ-FLAG band intensity (AU) Fig. S4. The UgtL and PhoQ proteins interact. Interaction between the UgtL-HA, PhoQ- FLAG, PhoZ-FLAG, EnvZ-FLAG proteins synthesized using an in vitro transcription-translation system. Immunoprecipitates were analyzed by Western blotting using antibodies recognizing the HA epitope and the FLAG epitope. Densitometry of each blot with ImageJ software is shown below the blots in the same order using arbitrary units (AU). Dashed lines in the densitometry graphs indicate signals from nonspecific binding of the UgtL-HA and FLAG-tagged proteins to antibodies recognizing the FLAG and HA epitopes, respectively.

6 AtpB PhoP-P PhoP Fig. S5. FLAG-tagged UgtL functions as WT UgtL in vivo. Phos-tag Western blot analysis of crude extracts prepared from Salmonella ugtl mutants (EG13682) harboring plasmids expressing the wild-type (pugtl) or FLAG-tagged form (pugtl-flag) of UgtL or the empty vector (puhe) grown under mildly acidic conditions. Samples were analyzed using antibodies recognizing PhoP and AtpB (loading control). Data are representative of three independent experiments, which produced similar results.

7 Fig. S6. PhoP activates transcription of the ugtl gene in mildly acidic ph and low Mg 2+ conditions. Abundance of ugtl transcripts in wild-type (WT, 14028s) and phop (MS7953s) Salmonella grown in N-minimal media with 1 mm of Mg 2+ at ph 4.9 (acidic ph), 10 µm Mg 2+ at ph 7.6 (low Mg 2+ ), or 1mM Mg 2+ at ph 7.6 (non-inducing conditions) to mid-log phase. The mean and SD from three independent experiments are shown. Unpaired students T-tests were performed between wild-type and isogenic ugtl mutant strains; ** p < 0.01, **** p < , n.s. p > 0.1.

8 Fig. S7. UgtL promotes Salmonella resistance to magainin 2 in a PhoP-dependent manner. Percentage survival of wild-type (WT, 14028s), phop (MS7953s) and ugtl (EG13682) Salmonella, and variations of the latter two strains harboring no plasmid (-), the empty vector (puhe), or a plasmid expressing UgtL (pugtl) grown under mildly acidic conditions and treated with magainin 2. The mean and SD from three independent experiments are shown. Unpaired students T-tests were performed between phop or ugtl Salmonella harboring puhe and those harboring pugtl; ** p < 0.01.

9 Table S1. Bacterial strains and plasmids used in this study. Strain or plasmid Description Reference Salmonella enterica 14028s MS5996s MS7953s EG13682 EG14943 wild-type phoq::tn10 phop::tn10 ugtl::kan repracement of the PhoP box with -35 hexamer phop- HA (28) (28) (28) (29) (21) JC804 hns-flag::cat This work JC805 hns-flag This work JC925 hns-flag ugtl:: kan This work JC969 hns-flag mgrb::cat This work JC1014 hns-flag phop*phoq::spec This work JC1015 repracement of the PhoP box with -35 hexamer phop- HA ugtl::kan This work JC1054 hns-flag mgrb::cat ugtl::kan This work JC1056 hns-flag phop*phoq::spec ugtl::kan This work JC1102 hns-flag phoqbongori (substitution with S. bongori phoq allele) This work JC1123 hns-flag phoqbongori (substitution with S. bongori This work

10 phoq allele) ugtl::kan Escherichia coli DH5α BTH101 F supe44 ΔlacU169 (f 80 laczδm15) hsdr17 reca1 enda1 gyra96 thi-1 rela1 F - cya-99 arad139 gale15 galk16 rpsl1 (Str R ) hsdr2 mcra1 mcrb1 (73) (44) Plasmids pcp20 reppsc101ts Ap R Cm R FLP + ci857 + (74) pkd3 pkd46 puhe21-2laci q pugtl repr6k Ap R FRT Cm R FRT reppsc101ts Ap R parabad γ β exo reppmb1 Ap R laci q reppmb1 Ap R laci q ugtl (74) (74) (43) (29) pugtl-flag reppmb1 Ap R laci q ugtl-flag This work pugtl-flag1-87 reppmb1 Ap R laci q ugtltruc-flag (1-87 amino acids) This work pugtl-flag22-87 reppmb1 Ap R laci q ugtltruc-flag (22-87 amino acids) This work pugtl-flag32-87 reppmb1 Ap R laci q ugtltruc-flag (32-87 amino acids) This work pugtl-flag60-87 reppmb1 Ap R laci q ugtltruc-flag (60-87 amino acids) This work pugtl-flag32-69 reppmb1 Ap R laci q ugtltruc-flag (32-69 amino acids) This work pugtl-flag1-69 reppmb1 Ap R laci q ugtltruc-flag (1-69 amino acids) This work pphoz reppmb1 Ap R laci q phoz This work

11 pkt25 put18 put18c KmR repp15a ApR reppmb1 ApR reppmb1 (75) (75) (75) pkt25-ugtl KmR repp15a ugtl This work pkt25-zip KmR repp15a zip (75) put18-phoq ApR reppmb1 phoq This work put18c-phoq ApR reppmb1 phoq This work put18-zip ApR reppmb1 zip (75) put18c-zip ApR reppmb1 zip This work pkt25-cigr KmR repp15a cigr This work

12 Table S2. Primers used in this study. Primers Sequence (from 5' to 3') for qrt-pcr 3023 CCA GCA GCC GCG GTA AT 3024 TTT ACG CCC AGT AAT TCC GAT T 6627 GGC GAC CGT AGT AAT ATC GAC AA 6628 CTT TCC TCC TGT TCA GCC TGT T 6962 GCA GGA GTA ATA TGT TGG ACA GTC AC 6963 GGG AGA TTG CTG CCC ACC 6964 AAA AGA TTA AAT CGG AGC GGG A 6965 TGA CGC TCC ATC CGC AAT A 7923 TTG AGT AAT ACC GCC AGC AGG 7924 AGA AAT CCA TCT GAC GCC GA W2033 W2034 CGG CGG ATC GCT GAC TTA T ATA GCG CCA CCG ACA GAG A for chromosomal mutations CAA TGG AAG AAC AAG GTA AGC AAC TGG AAG ATT TCC TGA TCA AGG AAG ACT ACA AGG ACG ACG ATG ACA AGT AAT GTA GGC TGG AGC TGC TTC G CAG GCA AAA AAA ATC CCG CCA GCG GCG GGA TTT TAA GCA TCC AGG AAG TAA ATA TGA ATA TCC TCC TTA GTT C

13 for in vitro protein synthesis GCG AAT TAA TAC GAC TCA CTA TAG GGC TTA AGT ATA AGG AGG AAA AAA T ATG AAG AAA TCA GAT GGT GAA ATT CAC GAA AAG AC AAA CCC CTC CGT TTA GAG AGG GGT TAT GCT AG TCA AGC GTA ATC TGG AAC ATC GTA TGG GTA CGG GCG TGA AGA AAC ATC CTG TGC CCC AAA TT GCG AAT TAA TAC GAC TCA CTA TAG GGC TTA AGT ATA AGG AGG AAA AAA T ATG AAT AAA TTT GCT CGC CAT TTT CTG CCG CTG TC AAA CCC CTC CGT TTA GAG AGG GGT TAT GCT AG TTA CTT GTC ATC GTC GTC CTT GTA GTC TTC CTC TTT CTG TGT GGG ATG CTG TCG GCC AA AAA CCC CTC CGT TTA GAG AGG GGT TAT GCT AG TTA CTT GTC ATC GTC GTC CTT GTA GTC TGC CTC TTT TGT CGT CCC CTG GAC GCG AGC CA GCG AAT TAA TAC GAC TCA CTA TAG GGC TTA AGT ATA AGG AGG AAA AAA T ATG AGG CGA ATG CGC TTC TCA CCG CGA AGT TCA TT for plasmid construction 2240 CGG GAT CCA GGA GGC TCA AAA TGA AGA AAT CAG 3143 CCC AAG CTT GTT GCA GCG GCG GAT CAC TTG TCA TCG TCG

14 TCC TTG TAG TCC GGG CGT GAA GAA ACA TCC TGT GCC CC ACT CTA GAG GAT CCC ATG AAT AAA TTT GCT TTA CTT AGG TAC CCG TTC CTC TTT CTG TGT CGA GGA GTC TAG AGA TGA AGA AAT CAG ATG GTG AAA TTC ACG AA GGT TGC AGG TAC CGA TCA CGG GCG TGA AGA AAC ATC CTG TGC CC GCC AAG CTT ATC ACT TAA GGA TAT GCC ATA AAT CAA GCG A GCC AAG CTT ATC ACT TGT CAT CGT CGT CCT TGT AGT CCT TAA GGA TAT GCC ATA AAT CAA GCG AAG CAT CGG GAT CCA GGA GGC TCA AAA TGT GGC TAA GGA AAT GTG GAC GGC TAT TAT TGC TGT TAC TTT CGG GAT CCA GGA GGC TCA AAA TGC TGT TAC TTT ACC GTT TCG TTA TCG GAT GGG CTT TTT TTC CGG GAT CCA GGA GGC TCA AAA TGT TTC ATC CCA TAA TAT TTG TAC AGA CTA TCG CAA TTA CTG GCC AAG CTT ATC ACT TGT CAT CGT CGT CCT TGT AGT CAG TCT GTA CAA ATA TTA TGG GAT GAA ATA ATA AGA CGC TCT AGA GAT GAA TAA TCG TCG TGG TTT AAC CGC CGT CCT GGC GAC CGC GGT ACC CGA TCA AAT ACG CCA TTA ATA ATC GCC GTG ACC AC

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