SUPPORTING FIGURE LEGENDS
FIG. S1. Activity of the HA-tagged FoxR proteins. The P. aeruginosa PAO1 pvdF strain (wildtype) and its isogenic foxR mutant bearing the pMPR8b plasmid (foxA::lacZ transcriptional fusion)
and either the pMMB67EH empty plasmid, pMMB/FoxR, pMMB/HA-FoxR or pMMB/FoxR-HA expressing
the indicated FoxR protein were grown under iron-restricted conditions without or with 1 µM
ferrioxamine. β-Galactosidase activity was measured as described in Experimental procedures.
FIG. S2. Processing of endogenous FoxR. (A) The P. aeruginosa PAO1 wild-type strain and its
isogenic HA-foxR mutant bearing the pMPR8b plasmid (foxA::lacZ transcriptional fusion) were grown
under iron-restricted conditions in the absence (-) and presence (+) of 1 µM ferrioxamine. βGalactosidase activity was measured as described in Experimental procedures. (B) The P. aeruginosa
PAO1 HA-foxR strain and its isogenic parent PAO1 (wild-type) were grown as above. Proteins were
detected on anti-HA immunoblot. Position of the protein fragments and the molecular size marker (in
kDa) are indicated.
FIG. S3. The role of DegP and DegS in the initial cleavage of FoxR. The pMMB/HA-FoxR
plasmid expressing the N-terminally HA-tagged P. aeruginosa FoxR protein (PaFoxR-NHA) was
expressed in the P. putida KT2440 wild-type strain and its isogenic ∆degP and ∆degS mutants grown
in low iron conditions supplemented with 1 mM IPTG. Proteins were detected using a monoclonal
anti-HA-tag antibody. Position of the protein fragments and the molecular size marker (in kDa) are
indicated.
FIG. S4. Determination of the FoxR initial cleavage site. (A) P. aeruginosa PAO1 cells bearing
the pMMB/FoxR-HA plasmid were grown to late log-phase in iron-restricted medium supplemented
with 1 mM IPTG, after which the 15 kDa FoxR C-domain was purified using anti-HA agarose.
Proteins were visualized on Coomassie-stained PVDF membranes, and confirmation was performed by
anti-HA immunoblot. The position of the C-domain is indicated, as well as the heavy and light chains
of the HA-antibody that co-eluted from the beads. (B) First 5 amino acids of C-FoxR as determined
by Edman degradation. The second residue (Arginine; R) could not be detected.
FIG. S5. Activity of ssFoxRperi-CHA. The P. aeruginosa PAO1 pvdF strain (wild-type) and its
isogenic foxR mutant bearing the pMPR8b plasmid (foxA::lacZ transcriptional fusion) and the
pMMB67EH
(empty),
pMMB/HA-FoxR,
pMMB/HA-FoxRN
or
pMMB/HA-FoxRN+ssFoxRperi-CHA
construct encoding the corresponding FoxR protein variant were grown under iron-restricted
conditions without or with 1 µM ferrioxamine. β-Galactosidase activity was determined as described in
Experimental procedures.
FIG. S6. Periplasmic localization of the FoxR C-domain. (A) The P. aeruginosa PAO1 foxR
mutant bearing the pMMB/FoxR-HA, pMMB/ssFoxRperi-CHA or pMMB/FoxRperi-CHA plasmid was
grown in iron-restricted medium supplemented with 1 mM IPTG. (B) The P. aeruginosa PAO1 foxR
bearing the pMMB/HA-FoxR, pMMB/FoxR-HA, pMMB/HA-FoxRN+ssFoxRperi-CHA, or pMMB/HAFoxRN+FoxRperi-CHA plasmid was grown in liquid CAS medium supplemented with 1 mM IPTG.
Protein samples were run under reducing (+DTT, left panel) or non-reducing (-DTT, right panel)
conditions. In both panels proteins were detected using a monoclonal anti-HA-tag antibody. The
position of the molecular size marker (in kDa) and the protein fragments are indicated.
FIG. S7. The GT cleavage site in other bacterial species. WebLogo of CSS anti-sigma factors
from the indicated species. See Table 1 for more information. Numbers indicate amino acid position in
the P. aeruginosa FoxR protein.
TABLE S1. Bacterial strains and plasmids used in this studya
Strain
Relevant characteristics
Reference
DH5
supE44 (lacZYA-argF)U169 80 lacZM15 hsdR17 (rK- mK+)
recA1 endA1 gyrA96 thi1 relA1; NalR
(Hanahan, 1983)
TOP10F’
F´lacIq, Tn10 (TetR) mcrA Δ(mrr-hsdRMS-mcrBC)
Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara leu) 7697 galU galK
rpsL (StrR) endA1 nupG; TcR
Invitrogen
PAO1
Wild-type strain
(Jacobs et al., 2003)
prc
Markerless PAO1 null mutant in the prc (PA3257) gene
(Bastiaansen et al., 2014)
rseP
Markerless PAO1 null mutant in the rseP (PA3649) gene
(Bastiaansen et al., 2014)
PAO1 HA-foxR
PAO1 in which the wild-type FoxR (PA2467) protein has been
replaced by an N-terminally HA-tagged version
This study
PAO1 pvdF
PAO1 isogenic parent strain of the foxR mutant. Contains the
insertion of a kanamycin cassette in the pvdF gene (pyoverdindeficient); KmR
(Mettrick and Lamont, 2009)
PAO1 pvdF foxR
PAO1 pvdF with a deletion of aminoacids 12-295 of the FoxR
protein; KmR
(Mettrick and Lamont, 2009)
KT2440
hsdR1, wild-type strain; RifR
(Franklin et al., 1981)
degP
degS
E. coli
P. aeruginosa
P. putida
Markerless KT2440 null mutant in the degP (PP1430) gene;
RifR
(Bastiaansen et al., 2014)
Markerless KT2440 null mutant in the degS (PP1301) gene;
RifR
(Bastiaansen et al., 2014)
Plasmid
pKNG101
Gene replacement suicide vector, oriR6K, oriTRK2, sacB; SmR
(Kaniga et al., 1991)
pKNG/HA-FoxR
pKNG101 carrying in XbaI-BamHI a 2.1-Kb PCR fragment
containing an N-terminally HA-tagged version and the regions
up- and downstream of the P. aeruginosa foxR (PA2467) gene;
SmR
This study
pMMB67EH
IncQ broad-host range plasmid, lacIq; ApR
(Fürste et al., 1986)
pMMB/FoxR
pMMB67EH carrying in EcoRI-XbaI a 0.99-Kb PCR fragment
containing the P. aeruginosa foxR (PA2467) gene; ApR
This study
pMMB/HA-FoxR
pMMB67EH carrying in EcoRI-XbaI a 1.02-Kb PCR fragment
containing a N-terminally HA-tagged P. aeruginosa foxR gene;
ApR
This study
pMMB/FoxR-HA
pMMB67EH carrying in EcoRI-XbaI a 1.02-Kb PCR fragment
containing a C-terminally HA-tagged P. aeruginosa foxR gene;
ApR
This study
pMMB/HA-FoxR-G191N
pMMB/HA-FoxR in which glycine-191 has been mutated to an
asparagine; ApR
This study
pMMB/HA-FoxR-T192Q
pMMB/HA-FoxR in which threonine-192 has been mutated to a
glutamine; ApR
This study
pMMB/HA-FoxR-T192A
pMMB/HA-FoxR in which threonine-192 has been mutated to an
alanine; ApR
This study
pMMB/HA-FoxR-R193K
pMMB/HA-FoxR in which arginine-193 has been mutated to a
lysine; ApR
This study
pMMB/FoxRperi-NHA
pMMB67EH carrying in EcoRI-XbaI a 0.67-Kb PCR fragment
encoding the periplasmic region of the P. aeruginosa FoxR
protein (residue 107 to 328) with an N-terminal HA-tag; ApR
This study
pMMB/FoxRperi-CHA
pMMB67EH carrying in EcoRI-XbaI a 0.67-Kb PCR fragment
encoding the periplasmic region of the P. aeruginosa FoxR
protein (residue 107 to 328) with a C-terminal HA-tag; ApR
This study
pMMB/HA-FoxRN
pMMB67EH carrying in EcoRI-XbaI a 0.58-Kb PCR fragment
encoding the first 191 amino acids of the P. aeruginosa FoxR
protein (the N-domain) with an N-terminal HA-tag; ApR
This study
pMMB/HA-FoxRN +
ssFoxRperi-CHA
pMMB/HA-FoxRN carrying in HindIII a 0.92-Kb PCR fragment
amplified from pTD-ssFoxRperi-CHA encoding the periplasmic
region of the P. aeruginosa FoxR protein with a C-terminal HAtag fused to the PelB signal sequence; ApR
This study
pMMB/HA-FoxRN +
FoxRperi-CHA
pMMB/HA-FoxRN carrying in HindIII a 0.81-Kb PCR fragment
amplified from pMMB/FoxRperi-CHA encoding the periplasmic
region of the P. aeruginosa FoxR protein with a C-terminal HAtag; ApR
This study
pMMB/HA-FiuR
pMMB67EH carrying in EcoRI-XbaI a 0.97-Kb PCR fragment
containing an N-terminally HA-tagged P. aeruginosa fiuR
(PA0471) gene; ApR
This study
pMMB/HA-FpvR
pMMB67EH carrying in KpnI-HindIII a 1.00-Kb PCR fragment
containing an N-terminally HA-tagged P. aeruginosa fpvR
(PA2388) gene; ApR
This study
pMMB/HA-FecR
pMMB67EH carrying in KpnI-HindIII a 0.95-Kb PCR fragment
containing an N-terminally HA-tagged P. aeruginosa fecR
(PA3900) gene; ApR
This study
pMMB/HA-FemR
pMMB67EH carrying in KpnI-HindIII a 0.95-Kb PCR fragment
containing an N-terminally HA-tagged P. aeruginosa femR
(PA1911) gene; ApR
This study
pMMB/HA-VreR
pMMB67EH carrying in EcoRI-XbaI a 0.99-Kb PCR fragment
containing an N-terminally HA-tagged P. aeruginosa vreR
(PA0676) gene; ApR
This study
pMMB/PPFiuR-NHA
pMMB67EH carrying in KpnI-XbaI a 0.95-Kb PCR fragment
containing an N-terminally HA-tagged P. putida fiuR (PP0351)
gene; ApR
This study
pMMB/PPFoxR-NHA
pMMB67EH carrying in EcoRI-XbaI a 0.95-Kb PCR fragment
containing an N-terminally HA-tagged P. putida foxR (PP0161)
gene; ApR
This study
pMMB/PPFoxR-NHA-GT
pMMB/PPFoxR-NHA in which glutamic acid-182 has been
mutated to a glycine and glycine-183 has been mutated to a
threonine; ApR
This study
pMPR8b
pMP220 carrying the P. aeruginosa foxA::lacZ transcriptional
fusion; TcR
(Llamas et al., 2006)
pTDpelB-CTwinStrep
RK2 based broad-host range expression plasmid including the 22
amino acid signal sequence of the Erwinia carotovora PelB
protein enabling export to the periplasmic environment; SmR
(Dammeyer et al., 2013)
pTD-ssFoxRperi-CHA
pTDpelB-CTwinStrep carrying in XbaI-HindIII a 0.67-Kb PCR
fragment encoding the periplasmic region of the P. aeruginosa
FoxR protein with a C-terminal HA-tag fused to the PelB signal
sequence; SmR
This study
a
ApR, KmR, NalR, RifR, SmR and TcR, resistance to ampicillin, gentamycin, kanamycin, nalidixic acid, rifampicin, streptomycin and tetracycline,
respectively
TABLE S2. Sequence of the primers used in this study
Amplified gene
Plasmid
Primer name
Primer sequence (5’  3’)a
pMMB/HA-FiuR
NHA-PA0471-E
AAAGAATTCATGTACCCGTACGACGTGCCGGACTACGCGTGCAGCG
CCGACGATCGTCATTCGC
PA0471Ra-X
TTTTCTAGATCAGCGTGGCACCACTTCGAACC
NHA-PA0676-E
AAAGAATTCATGTACCCGTACGACGTGCCGGACTACGCGTGCACAG
CCTCAGACTCCGCCGCC
PA0676Ra-X
TTTTCTAGATCAGCCGAGCAGCACCACCCCGCCCGG
PA1911HAF-K
AAAGGTACCATGTACCCGTACGACGTGCCGGACTACGCGAACGCC
GCGGACGAAGGC
PA1911R-H
TTTAAGCTTTCACGCCGGCACCAGGCTGATCC
PA2388HAF-K
AAAGGTACCATGTACCCGTACGACGTGCCGGACTACGCGAAGACAC
CCTCTCCCGCC
PA2388R-H
TTTAAGCTTATCAGAGCTGGGCGTAACGGC
FoxIF-X
AAATCTAGAGCTGGACTGGGAAGACGTGC
HA-FoxRR
CGCGTAGTCCGGCACGTCGTACGGGTACATGGCTCCGCTCATTCGC
CGT
HA-FoxRF
ATGTACCCGTACGACGTGCCGGACTACGCGGACGGGACGCGCGGTA
FoxRR-B
AAAGGATCCGAGGACGACGGCGGTGGCG
PA2467Fa-E
AAAGAATTCGTGGACGGGACGCGCGGTAGGGTC
PA2467Ra-X
TTTTCTAGATCAGGCGGCGACCACCCTCAC
NHA-PA2467-E
AAAGAATTCATGTACCCGTACGACGTGCCGGACTACGCGTGCGACG
GGACGCGCGGTAGGGTC
PA2467Ra-X
TTTTCTAGATCAGGCGGCGACCACCCTCAC
PA2467Fa-E
AAAGAATTCGTGGACGGGACGCGCGGTAGGGTC
CHA-PA2467-X
TTTTCTAGATTAGCACGCGTAGTCCGGCACGTCGTACGGGTAGGC
GGCGACCACCCTCACCCACCAG
NHA-PA2467-E
AAAGAATTCATGTACCCGTACGACGTGCCGGACTACGCGTGCGACG
GGACGCGCGGTAGGGTC
PA2467R-GN
CGTTGAAACGGGTGTTGAGGGCC
PA2467F-GN
CTGTTCGAGGCCCTCAACACCC
PA2467Ra-X
TTTTCTAGATCAGGCGGCGACCACCCTCAC
NHA-PA2467-E
AAAGAATTCATGTACCCGTACGACGTGCCGGACTACGCGTGCGACG
GGACGCGCGGTAGGGTC
PA2467R-TQ
GTACGTTGAAACGTTGGCCGA
PA2467F-TQ
CGAGGCCCTCGGCCAACGTTTC
PA2467Ra-X
TTTTCTAGATCAGGCGGCGACCACCCTCAC
NHA-PA2467-E
AAAGAATTCATGTACCCGTACGACGTGCCGGACTACGCGTGCGACG
GGACGCGCGGTAGGGTC
PA2467R-TA
GTACGTTGAAACGGGCGCCGA
PA2467F-TA
CGAGGCCCTCGGCGCCCGTTTC
PA2467Ra-X
TTTTCTAGATCAGGCGGCGACCACCCTCAC
NHA-PA2467-E
AAAGAATTCATGTACCCGTACGACGTGCCGGACTACGCGTGCGACG
GGACGCGCGGTAGGGTC
PA2467R-RK
CGTACGTTGAATTTGGTGCC
P. aeruginosa PAO1
fiuR (PA0471)
vreR (PA0676)
femR (PA1911)
fpvR (PA2388)
foxR (PA2467)
pMMB/HA-VreR
pMMB/HA-FemR
pMMB/HA-FpvR
pKNG-HA-FoxR
pMMB/FoxR
pMMB/HA-FoxR
pMMB/FoxR-HA
pMMB/HA-FoxRG191N
pMMB/HA-FoxRT192Q
pMMB/HA-FoxRT192A
pMMB/HA-FoxRR193K
GGG
PA2467F-RK
GCCCTCGGCACCAAATTCAAC
PA2467Ra-X
TTTTCTAGATCAGGCGGCGACCACCCTCAC
PA2467periNHAF-E
AAAGAATTCATGTACCCGTACGACGTGCCGGACTACGCGTGCGATA
GCCTGCCCTGGCAGCGC
PA2467Ra-X
TTTTCTAGATCAGGCGGCGACCACCCTCAC
pMMB/FoxRperiCHA
PA2467periF-E
AAAGAATTCATGGATAGCCTGCCCTGGCAGCGC
CHA-PA2467-X
TTTTCTAGATTAGCACGCGTAGTCCGGCACGTCGTACGGGTAGGC
GGCGACCACCCTCACCCACCAG
pMMB/HAFoxRN
NHA-PA2467-E
AAAGAATTCATGTACCCGTACGACGTGCCGGACTACGCGTGCGACG
GGACGCGCGGTAGGGTC
PA2467RN2-X
TTTTCTAGATCAGCCGAGGGCCTCGAACAGCC
pMMB/HAFoxRN +
ssFoxRperi-CHA
pTD-H1
TTTAAGCTTCTGAAATGAGCTGTTGACAATTAATC
pTD-R
GATCTATCAACAGGAGTCCAAGAC
pMMB/HAFoxRN +
FoxRperi-CHA
MMBF1-HindIII
TTTAAGCTTGCGCCGACATCATAACGGTTC
MMB67-3’
TGTTTTATCAGACCGCTTC
PURE DNA
template
FoxRperi-NHA
FoxRperiNHA-F
GCGAATTAATACGACTCACTATAGGGCTTAAGTATAAGGAGGAAAA
AATATGTACCCGTACGACGTGCCGGACTAC
FoxRperiNHA-R
AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATCAGGCGGCGA
CCACCCTCAC
PURE DNA
template
FoxRperi-CHA
FoxRperiCHA-F
GCGAATTAATACGACTCACTATAGGGCTTAAGTATAAGGAGGAAAA
AATATGGATAGCCTGCCCTGGCAGCGC
FoxRperiCHA-R
AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATTAGCACGCGT
AGTCCGGCACGTC
PURE DNA
template
FoxRperi-CHAT192Q
FoxRperiCHA-F
GCGAATTAATACGACTCACTATAGGGCTTAAGTATAAGGAGGAAAA
AATATGGATAGCCTGCCCTGGCAGCGC
PA2467R-TQ
GTACGTTGAAACGTTGGCCGA
PA2467F-TQ
CGAGGCCCTCGGCCAACGTTTC
FoxRperiCHA-R
AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATTAGCACGCGT
AGTCCGGCACGTC
PURE DNA
template
FoxRperi-CFLAG
FoxRperiCHA-F
GCGAATTAATACGACTCACTATAGGGCTTAAGTATAAGGAGGAAAA
AATATGGATAGCCTGCCCTGGCAGCGC
FoxRperiCflag-R
AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTACTTGTCGTCATC
GTCTTTGTAGTCGGCGGCGACCACCC
PURE DNA
template
FoxRperiNHA+CFLAG
FoxRperiNHA-F
GCGAATTAATACGACTCACTATAGGGCTTAAGTATAAGGAGGAAAA
AATATGTACCCGTACGACGTGCCGGACTAC
FoxRperiCflag-R
AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTACTTGTCGTCATC
GTCTTTGTAGTCGGCGGCGACCACCC
pTD-ssFoxRperiCHA
PA2467periF-X
AAATCTAGAGATAGCCTGCCCTGGCAGCGC
FoxRCHAR-H
TTTAAGCTTAGCACGCGTAGTCCGGCA
pMMB/HA-FecR
PA3900HAF-K
AAAGGTACCATGTACCCGTACGACGTGCCGGACTACGCGAGCCTGC
CCGCCGCACC
PA3900R-H
TTTAAGCTTCATTCGCGCGGGTGCACGCC
NHA-PP0161-E
AAAGAATTCATGTACCCGTACGACGTGCCGGACTACGCGTGCACCG
TCGCACGCCCTGATAC
PP0161Ra-X
TTTTCTAGATCAGGTGATGCTCTCTACATGC
pMMB/FoxRperiNHA
fecR (PA3900)
P. putida KT2440
foxR (PP0161)
pMMB/PPFoxRNHA
pMMB/PPFoxRNHA-GT
fiuR (PP0351)
a
pMMB/PPFiuRNHA
NHA-PP0161-E
AAAGAATTCATGTACCCGTACGACGTGCCGGACTACGCGTGCACCG
TCGCACGCCCTGATAC
PP0161R-GT
GGCCACAAAGCGTGTTCCGAAGCC
PP0161F-GT
CTGGAAGGCTTCGGAACACGCTTT
PP0161Ra-X
TTTTCTAGATCAGGTGATGCTCTCTACATGC
NHA-PP0351-K
AAAGGTACCATGTACCCGTACGACGTGCCGGACTACGCGTGCAGCA
ACCTCCCGGTTTCGTCCC
PP0351Ra-X
TTTTCTAGACTATTTGGAAGTGACGTTCACCC
The sequences of the restriction sites are indicated in bold and the annealing region is underlined
REFERENCES FOR SUPPORTING INFORMATION
Bastiaansen, K.C., Ibañez, A., Ramos, J.L., Bitter, W., and Llamas, M.A. (2014) The Prc and RseP proteases
control bacterial cell-surface signalling activity. Environ Microbiol 16: 2433-2443.
Dammeyer, T., Timmis, K., and Tinnefeld, P. (2013) Broad host range vectors for expression of proteins with
(Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein. Microbial Cell Factories
12: 49.
Franklin, F.C.H., Bagdasarian, M., Bagdasarian, M.M., and Timmis, K.N. (1981) Molecular and functional analysis
of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring
meta-cleavage pathway. Proc Natl Acad Sci U S A 78: 7458-7462.
Fürste, J.P., Pansegrau, W., Frank, R., Blöcker, H., Scholz, P., Bagdasarian, M., and Lanka, E. (1986) Molecular
cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene 48: 119-131.
Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166: 557-580.
Jacobs, M.A., Alwood, A., Thaipisuttikul, I., Spencer, D., Haugen, E., Ernst, S. et al. (2003) Comprehensive
transposon mutant library of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 100: 14339-14344.
Kaniga, K., Delor, I., and Cornelis, G.R. (1991) A wide-host-range suicide vector for improving reverse genetics in
Gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene 109: 137-141.
Llamas, M.A., Sparrius, M., Kloet, R., Jimenez, C.R., Vandenbroucke-Grauls, C., and Bitter, W. (2006) The
heterologous siderophores ferrioxamine B and ferrichrome activate signaling pathways in Pseudomonas
aeruginosa. J Bacteriol 188: 1882-1891.
Mettrick, K.A., and Lamont, I.L. (2009) Different roles for anti-sigma factors in siderophore signalling pathways
of Pseudomonas aeruginosa. Mol Microbiol 74: 1257-1271.