SUPPLEMENTAL FIGURE LEGENDS
FIG. S1. PP2192 sequence analyses. (A) Conserved domains detected in the PP2192 protein. ( B)
PP2192 protein sequence and motifs prediction. The predicted cytoplasmic sigma domain (aminoacids 1169) is depicted in red with the domains 2 and 4 characteristic of ECF sigma factors underlined and
double underlined, respectively. The TM domain (aminoacids 170-186) is shown in grey, and the
predicted periplasmic anti-sigma domain (aminoacids 187-374) in blue. The length of the different K1 to
K3 constructs is indicated in the sequence as well as the insertion point of the miniTn5-Km in the iutY-Tn5
mutant. (C) Alignment of the PP2192 (IutY) N-terminal domain with the E. coli FecI, P. aeruginosa FoxI
and P. putida PupI ECF sigma factors. The PP2192 N-domain contains the domains 2 and 4 characteristic
of sigma factors belonging to the ECF subfamily (shaded). (D) Alignment of the PP2192 (IutY) C-terminal
domain with the E. coli FecR, P. aeruginosa FoxR and P. putida PupR anti-sigma factors. The PP2192 Cdomain resembles the C-terminal domain of TM anti-sigma factors but lacks the N-terminal cytoplasmic
domain (first 100 aminoacids), which is the domain of (normal) anti-sigma factors that binds the sigma
factor. The PP2192 TM domain is shaded.
FIG. S2. PP2192 promoter region. Sequence of the putative FUR box in the PP2192 (iutY) promoter
region is surrounded. Residues matching with the FUR box consensus sequence (below) are indicated in
bold. The PP2192 start codon and the putative -10 and -35 regions are indicated.
FIG. S3. Southern-blot analyses of the iutY-Tn5 and iutA-Tn5 mutants. The restriction map of
both the P. putida iutY-Tn5 and iutA-Tn5 chromosomal region at the point of the miniTn5-Km transposon
insertion is shown. The kanamycin resistance gene was used as a probe to determine the size of the
BamHI and PstI, respectively, chromosomal fragments. The sizes of such fragments are indicated in the
scheme. For the Southern blot analyses, total DNA was prepared from P. putida KT2440 (wild-type, WT),
and the iutY-Tn5 and iutA-Tn5 mutants, and digested either with BamHI or with PstI. After
electrophoresis on agarose gel, the digested DNA was transferred to a nylon hybridization membrane. The
probe was labelled with digoxigenin-dUTP (Roche). For DNA prehybridization and hybridization, highstringency conditions (42ºC, 50% [v/v] formamide) were used. The digoxigenin-labelled hybrid DNA was
detected by using an enzyme immunoassay according to the manufacturer’s instructions (Roche).
FIG. S4. Screening to identify proteases involved in the P. putida aerobactin-mediated CSS
pathway. The P. putida KT2440 wild-type strain and its isogenic null degP and degS mutants (A) and
miniTn5-Km mutants (B) bearing the pMPK4 plasmid (iutA::lacZ fusion) were grown under iron-restricted
conditions without or with aerobactin containing supernatant. -galactosidase was measured as described
in Experimental procedures.
FIG. S5. Activity of the HA-tagged IutY proteins. The P. putida iutY mutant bearing the pMPK4
plasmid (iutA::lacZ fusion) and the indicated pMMB67EH-derivative plasmids, in which the inserted gene
product was or not HA-tagged, were grown under iron-restricted conditions with aerobactin containing
supernatant. -galactosidase activity was measured as described in Experimental procedures.
Fig. S6. Activity of the P. putida ferrioxamine- and ferrichrome-mediated CSS pathways. (A)
Activity of the ferrioxamine- and ferrichrome-mediated CSS systems in the P. putida degP and degS
mutants, and (B) complementation of the prc and rseP mutations for the ferrichrome induced pathway.
The indicated strains were grown under iron-restricted conditions without or with the siderophores. galactosidase was measured as described in Materials and Methods.
Fig. S7. Model for regulated intramembrane proteolysis (RIP) in the activation of stressresponsive ECF sigma factors. (A) Activation of the B. subtilis ECF sigma factor σW. In unstressed
cells, σW is bound to the RsiW anti-sigma factor and inactive. In response to stress, the site-1 protease
PrsW cleaves in the C-terminal end of the periplasmic domain of RsiW. Subsequently, periplasmic bulk
proteases like Prc are required for trimming the rest of the C-terminal domain of the anti-sigma factor and
for producing the substrate for the site-2 protease RasP (RseP homologue). RasP cleaves in the TM region
of RsiW and the remaining cytosolic portion of the protein is degraded by cytosolic proteases such as
ClpXP. Consequently, σW is free to associate with the RNAP core enzyme and drive transcription of the σW
regulon. (Figure adapted from Heinrich et al., 2009) (B) Activation of the E. coli ECF sigma factor σE. In
unstressed cells, σE is inactive through its interaction with the RseA anti-sigma factor. The DegS and RseP
proteases are inactive through inhibitory interactions with their own PDZ domains, RseB that binds and
protects the periplasmic domain of RseA, and a glutamine-rich region of RseA. In stressed cells, unfolded
proteins accumulate in the periplasm and activate the DegS protease by binding its PDZ domain. RseB is
in this situation displaced from the RseA protein and DegS cleaves the periplasmic domain of the antisigma factor. This site-1 cleavage removes the inhibitory interaction between RseP and RseA, and enables
RseP to cleave in the TM region of RseA. The remainder of RseA is degraded in the cytosol by ClpXP
proteases, upon which σE is free to interact with the RNAP core enzyme and direct transcription of its
target genes. The σE regulon mainly encodes proteins that are involved in the restoration of the proper
folding of outer membrane proteins. The P. aeruginosa σE, RseA, RseB, DegS and RseP homologues (σAlgT,
MucA, MucB, AlgW and MucP, respectively) are indicated between brackets. CW, cell wall; OM, outer
membrane; P, periplasm; CM, cytoplasmic membrane; C, cytoplasm.
TABLE S1. Bacterial strains and plasmids used in this studya
Strains or plasmid
Relevant characteristics
Reference
E. coli
C600
F- tonA21 thi-1 thr-1 leuB6 lacY1 glnV44 rfbC1 fhuA1 λ-; RifR
(Hanahan 1983)
CC118pir
∆(ara-leu) araD ∆lacX74 galE galK phoA20 thi-1 rpsE rpoB argE
recA1, lysogenized with pir; RifR
(Herrero et al. 1990)
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
This study
rseP
Markerless PAO1 null mutant in the rseP (PA3649) gene
This study
KT2440
hsdR1, wild-type strain; RifR
(Franklin et al. 1981)
iutA-Tn5
KT2440 carrying a miniTn5-Km in the iutA (PP2193) gene
(insertion after codon 438); RifR, KmR
(Molina-Henares et al. 2010)
and this study
iutY-Tn5
KT2440 carrying a miniTn5-Km in the iutY (PP2192) gene
(insertion after codon 256); RifR, KmR
(Molina-Henares et al. 2010)
and this study
PP0160-Tn5
KT2440 carrying a miniTn5-Km in the foxA gene (insertion after
codon 205); RifR, KmR
(Molina-Henares et al. 2010)
and this study
PP0350-Tn5
KT2440 carrying a miniTn5-Km in the fiuA gene (insertion after
codon 253); RifR, KmR
(Molina-Henares et al. 2010)
and this study
PP0625-Tn5 (1)
KT2440 carrying a miniTn5-Km in the clpB gene (insertion after
codon 279); RifR, KmR
(Molina-Henares et al. 2010)
and this study
PP0625-Tn5 (2)
KT2440 carrying a miniTn5-Km in the clpB gene (insertion after
codon 610); RifR, KmR
(Molina-Henares et al. 2010)
and this study
PP0680-Tn5
KT2440 carrying a miniTn5-Km in a gene encoding a putative
cytoplasmic protease (insertion after codon 403); RifR, KmR
(Molina-Henares et al. 2010)
and this study
PP1321-Tn5
KT2440 carrying a miniTn5-Km in the sspB gene (insertion after
codon 148); RifR, KmR
(Molina-Henares et al. 2010)
and this study
PP1443-Tn5 (1)
KT2440 carrying a miniTn5-Km in the lon-1 gene (insertion after
codon 11); RifR, KmR
(Molina-Henares et al. 2010)
and this study
PP1443-Tn5 (2)
KT2440 carrying a miniTn5-Km in the lon-1 gene (insertion after
codon 105); RifR, KmR
(Molina-Henares et al. 2010)
and this study
PP1719-Tn5
KT2440 carrying a miniTn5-Km in the prc gene (insertion after
codon 624); RifR, KmR
(Molina-Henares et al. 2010)
and this study
PP2302-Tn5
KT2440 carrying a miniTn5-Km in the lon-2 gene (insertion after
codon 615); RifR, KmR
(Molina-Henares et al. 2010)
and this study
PP2725-Tn5
KT2440 carrying a miniTn5-Km in the pfpI gene (insertion after
codon 18); RifR, KmR
(Molina-Henares et al. 2010)
and this study
PP3922-Tn5
KT2440 carrying a miniTn5-Km in a putative periplasmic protease
(Molina-Henares et al. 2010)
P. aeruginosa
P. putida
(insertion after codon 107); RifR, KmR
and this study
PP4008-Tn5(1)
KT2440 carrying a miniTn5-Km in the clpA gene (insertion after
codon 80); RifR, KmR
(Molina-Henares et al. 2010)
and this study
PP4008-Tn5(2)
KT2440 carrying a miniTn5-Km in the clpA gene (insertion after
codon 128); RifR, KmR
(Molina-Henares et al. 2010)
and this study
PP5001-Tn5
KT2440 carrying a miniTn5-Km in the hslU gene (insertion after
codon 295); RifR, KmR
(Molina-Henares et al. 2010)
and this study
PP5058-Tn5
KT2440 carrying a miniTn5-Km in a putatative C-terminal
protease (insertion after codon 62); RifR, KmR
(Molina-Henares et al. 2010)
and this study
degP
Markerless KT2440 null mutant in the degP (PP1430) gene; RifR
This study
degS
Markerless KT2440 null mutant in the degS (PP1301) gene; RifR
This study
iutY
Markerless KT2440 null mutant in the iutY (PP2192) gene;
This study
prc
Markerless KT2440 null mutant in the prc (PP1719) gene;
rseP
Markerless KT2440 null mutant in the rseP (PP1598) gene; RifR
This study
ColV-K30
Plasmid containing the aerobactin biosynthetic pathway
(de Lorenzo et al. 1986)
pBBR1MCS-5
oriTRK2; GmR
(Kovach et al. 1995)
pBBR-PPprc
pBBR1MCS-5 carrying in BamHI a 2.6-Kb PCR fragment
containing the P. putida prc (PP1719) gene; GmR
This study
pBBR-PPrseP
pBBR1MCS-5 carrying in XhoI-HindIII a 1.6 Kb PCR fragment
containing the P. putida rseP (PP1598) gene; GmR
This study
pBSL141
Source of the Gm cassette; ApR, GmR
(Alexeyev et al. 1995)
pKNG101
Gene replacement suicide vector, oriR6K, oriTRK2, sacB;
pKdegP
pKNG101 carrying in XbaI-BamHI a 2.1-Kb PCR fragment
containing the regions up- and downstream the P. putida degP
(PP1430) gene; SmR
This study
pKdegS
pKNG101 carrying in XbaI-BamHI a 2.3-Kb PCR fragment
containing the regions up- and downstream the P. putida degS
(PP1301) gene; SmR
This study
pKiutY
pKNG101 carrying in XbaI-BamHI a 2.3-Kb PCR fragment
containing the regions up- and downstream the P. putida iutY
(PP2192) gene; SmR
This study
pKprc
pKNG101 carrying in XbaI-BamHI a 2.6-Kb PCR fragment
containing the regions up- and downstream the P. putida prc
(PP1719) gene; SmR
This study
pKrseP
pKNG101 carrying in XbaI-BamHI a 1.9-Kb PCR fragment
containing the regions up- and downstream the P. putida rseP
(PP1598) gene; SmR
This study
pKPAprc
pKNG101 carrying in XbaI-BamHI a 2.1-Kb PCR fragment
containing the regions up- and downstream the P. aeruginosa
prc (PA3257) gene; SmR
This study
pKPArseP
pKNG101 carrying in XbaI-BamHI a 1.8-Kb PCR fragment
containing the regions up- and downstream the P. aeruginosa
rseP (PA3649) gene; SmR
This study
pMMB67EH
IncQ broad-host range plasmid, lacIq; ApR
(Fürste et al. 1986)
RifR
RifR
This study
Plasmids
SmR
(Kaniga et al. 1991)
pMMB-iutA
pMMB67EH carrying in EcoRI-HindIII a 2.7-Kb PCR fragment
containing the P. putida iutA (PP2193) gene; ApR
This study
pMMBK1
pMMB67EH carrying in EcoRI-HindIII a 1.7-Kb PCR fragment
containing the P. putida iutY (PP2192) gene; ApR
This study
pMMBK2
pMMB67EH carrying in EcoRI-HindIII a 0.92-Kb PCR fragment
containing a partial P. putida iutY gene (aminoacids 1-171); ApR
This study
pMMBK3
pMMB67EH carrying in EcoRI-HindIII a 0.99-Kb PCR fragment
containing a partial P. putida iutY gene (aminoacids 1-194); ApR
This study
pMMBK1-HA
pMMBK1 in which the iutY gene has been N-terminally HAtagged; ApR
This study
pMMBK2-HA
pMMBK2 in which the iutY K2 fragment gene has been Nterminally HA-tagged; ApR
This study
pMMBK3-HA
pMMBK3 in which the iutY K3 fragment gene has been Nterminally HA-tagged; ApR
This study
pMP220
IncP broad-host-range lacZ fusion vector; TcR
(Spaink et al. 1987)
pMPK4
pMP220 carrying in EcoRI-BamHI the P. putida iutA (PP2193)
promoter region cloned upstream the lacZ gene; TcR
This study
pMPFiuA
pMP220 carrying in EcoRI-BamHI the P. aeruginosa fiuA
(PA0470) promoter region cloned upstream the lacZ gene; TcR
(Llamas et al. 2006)
pMPR8b
pMP220 carrying in EcoRI-BamHI the P. aeruginosa foxA
(PA2466) promoter region cloned upstream the lacZ gene; TcR
(Llamas et al. 2006)
pMP-PPfoxA
pMP220 carrying in EcoRI-BamHI the P. puitda foxA (PP0160)
promoter region cloned upstream the lacZ gene; TcR
This study
pMP-PPfiuA
pMP220 carrying in EcoRI-BamHI the P. putida fiuA (PP0350)
promoter region cloned upstream the lacZ gene; TcR
This study
pRK600
Helper plasmid, oriColE1, mobRK2, traRK2; CmR
(de Lorenzo and Timmis
1994)
a
ApR, GmR, 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 (or deleted)
gene and promoter region
Plasmid
Name
Sequence (5’  3’)a
pKΔPAprc
PA3256F-X
AAATCTAGAGCCAGACCTTCAACCCCAG
ΔPAprcR-E
CCAGGAATTCTGGGCGCTG
ΔPAprcF-E
TAAGAATTCACTGAGTTCAGCGGGAGCG
PA3258R-B
TAAGGATCCGATGGACGATGCCGATGGG
PA3650F-X
AACTCTAGATCCTCTTGACCGCCTCCG
ΔPArsePR-E
AAAGAATTCGTGGAACGTCACCAGC
ΔPArsePF-E
GAGTCGTCTGTAGTCATGTTGAATTCG
PA3648R-B
AATGGATCCGGTGTAGCCCTCGTTGCCC
Pr-PP0160F-E
AATGAATTCTGCTACCGCAGACGTTGCC
Pr-PP0160R-X
TGCTCTAGATTCGGGCTGAAAGTGTGGG
Pr-PP0350F-E
AAAGAATTCGGCATACGCAGTGGTGGG
Pr-PP0350R-X
AACTCTAGATATGATCAACGGCACGGG
PP1303F-X
AACTCTAGATTCCTCGACCATCTTGTCGC
ΔdegSR-E
AATGAATTCTGGCAGTGGGGCATGAGCG
ΔdegSF-E
AAAGAATTCGCCAGGAAGAGAAATAAACCC
PP1300R-B
AAAGGATCCGAGGGCTACGTGTTCGC
rseBF-X
AAATCTAGACTCAAGGGCACTGAAACCG
degPR-E
AAAGAATTCAAAGACCCCTGCACAACGC
degPF-E
CCAGAATTCAATAAGCAGTTTCGCAAGGC
PP1431R-B
TTAGGATCCAGCGAGGAGTCGTTCAGGG
PP1598F-X
AAACTCGAGATCGCGGGTATGATCGAACAGGTA
PP1598R-H
TTTAAGCTTTGGCAATAAACAACCTGCCGTCAC
PP1598F-X
TATTCTAGATCCTGCTTACCGCGTCCG
PP1598R-E
AACGAATTCCGCTGTCATGTCCATCTCCG
PP1598F-E
ATAGAATTCATAGGGGTGATGTTGCTCGC
PP1598R-B
TATGGATCCTCTTCGGTCTTGGTGTTGCC
PP1719F-X
AAATCTAGAGCAAACGCGGTCACCCACG
PP1719R-B
TTTGGATCCCCCGGCCCTCATATTTCACG
PP1720F-X
GAATCTAGACGAGGAATGACTTGCCGTT
ΔPP1719R-E
TTAGAATTCATTGATCACGCATAGTAGGC
ΔPP1719F-E
AAAGAATTCAAGAAGTAAGGCACCTCAGC
PP1718R-B
TATGGATCCTACTGGCTTCTTTGAGTGCG
P. aeruginosa PAO1
PA3257 (prc)
PA3649 (rseP)
pKΔPArseP
P. putida KT2440
PP0160 (foxA)
PP0350 (fiuA)
PP1301 (degS)
PP1430 (degP)
PP1598 (rseP)
pMP-PPfoxA
pMP-PPfiuA
pKΔdegS
pKΔdegP
pBBR-PPrseP
pKΔrseP
PP1719 (prc)
pBBR-PPprc
pKΔprc
PP2192 (iutY)
pMMBK1
pMMBK2
pMMBK3
pMMBK1-HA
pMMBK2-HA
pMMBK3-HA
pKΔiutY
PP2193 (iutA)
pMMB-iutA
pMPK4
a
PP_2192F-E
AACGAATTCTGCCCCTGCTGGTCACTGAC
PP_2192R-H(1)
GAGAAGCTTAAGTCGTAAATGAGAATGGG
PP_2192F-E
AACGAATTCTGCCCCTGCTGGTCACTGAC
PP_2192R-H(3)
GATAAGCTTCAACCCGCCTGCTTCAGCC
PP_2192F-E
AACGAATTCTGCCCCTGCTGGTCACTGAC
PP_2192R-H(2)
TCTAAGCTTCAGGCCAGCAGTATCGGGGC
NHA-PP2192-E
AAAGAATTCATGTACCCGTACGACGTGCCGGACTACGC
GTGCCTGACTTCACCCATGTCGGGCC
PP2192R-X
TTTTCTAGATCAATGCACCACGGCCAACCACGGCAG
NHA-PP2192-E
AAAGAATTCATGTACCCGTACGACGTGCCGGACTACGC
GTGCCTGACTTCACCCATGTCGGGCC
PP_2192R-H(3)
GATAAGCTTCAACCCGCCTGCTTCAGCC
NHA-PP2192-E
AAAGAATTCATGTACCCGTACGACGTGCCGGACTACGC
GTGCCTGACTTCACCCATGTCGGGCC
PP_2192R-H(2)
TCTAAGCTTCAGGCCAGCAGTATCGGGGC
PP2190F-X
TAATCTAGAACTCGTCCTCGTCAATGGG
ΔPP2192R-E
ATAGAATTCAGCATGATTGGGGAACAGCC
ΔPP2192F-E
ATTGAATTCCTGTTCCGGCCTCTTAGC
PP2193R-B
GTTGGATCCTAGATACCCGTGCTGCCG
PRPP_2193F
TTAGAATTCATTGATCAGCCTGTTCCG
PP2193R-H
CCTAAGCTTGCCACCACAATCAGTAAGCC
PRPP_2193F
TTAGAATTCATTGATCAGCCTGTTCCG
PRPP_2193R
GGATCTAGACAAGTCGTAAATGAGAATGG
The sequences of the restriction sites are indicated in bold and the annealing region is underlined
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