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JB00458-09v2

Supplemental Material

Role of periplasmic chaperones and BamA(YaeT)-complex for folding

and secretion of Intimin from enteropathogenic E. coli strains

by

Gustavo Bodelón, Elvira Marín and Luis Ángel Fernández*

Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior

de Investigaciones Científicas (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain.

*For correspondence: lafdez@cnb.csic.es

Supplemental Methods

Construction of mutant E. coli strains

To obtain the null and conditional mutant derivatives of E. coli UT5600, the gene replacement

procedure with PCR fragments was employed (1). The kanamycin (KmR) resistant gene

cassette, flanked by FRT recombination sites, was amplified by PCR from pKD4 (ApR)

plasmid (1) using specific oligonucleotides flanking the 5'- and 3'-ends of the coding sequence

of each targeted gene, as described below. The zeoRExBAD (zeoR araC PBAD) promoter

cassette was amplified from the chromosome of strain E. coli TG1zeoRExBAD (a gift of Dr.

Jean-Marc Ghigo) using specific oligonucleotides hybridizing with upstream promoter region

of bamA or surA genes (5'-primer) and the beginning of their coding sequence (3'-primer), as

described below. The zeoRExBAD (zeoR araC PBAD) promoter cassette is a modification of

the catRExBAD cassette (3). All PCR reactions were performed with Vent DNA polymerase

(New England Biolabs). Custom oligonucleotides were synthesized and PAGE-purified

(Sigma-Genosys).

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E. coli UT5600 ΔaraC (UT5601) strain:

The oligonucleotides AraC KO1 (5’- CCC TAT GCT ACT CCG TCA AGC CGT CAA TTG

TCT GAT TCG TTA Cgt gta ggc tgg agc tgc ttc) and AraC KO2 (5’- CCG CCA AAG CTC

GCA CAG AAT CAC TGC CAA AAT CGA GGC Cat atg aat atc ctc ctt agt) were used as

primers for PCR on pKD4 template. The PCR product was electroporated into E. coli UT5600

carrying pKD46 (1). The araC::kan genotype was verified by PCR using the oligonucleotides

AraC2 (5’- CTG GTG GCG ATC TCT TCA CCG GTA GC) and k2 (5’- CGG TGC CCT

GAA TGA ACT GC). The KmR resistant gene was removed by Flipase-mediated

recombination after transformation with pCP20 (1).

E. coli UTdegP (UT5601 degP::kan) strain:

The UTdegP strain was obtained by transformation of E. coli UT5601 carrying pKD46 with

the PCR product obtained after amplification of template pKD4 with the oligonucleotides

DegP KO3 (5’- CAC AGC AAT TTT GCG TTA TCT GTT AAT CGA GAC TGA AAT Agt

gta ggc tgg agc tgc ttc) and DegP KO4 (5’- GAA GAT GTA TGG AGT TGT GGT GAA

GTT CAC AGA TTG TAA Gat atg aat atc ctc ctt agt). The degP::kan genotype was verified

by PCR using the oligonucleotides DegP3 (5’- GCA GAA ACT TTA GTT CGG AAC TTC)

and kt (5’- CGG CCA CAG TCG ATG AAT CC).

E. coli UTskp (UT5600 skp::kan) strain:

The UTskp strain was obtained by transformation of E. coli UT5600 strain carrying pKD46

with the PCR product obtained after amplification of template pKD4 with the

oligonucleotides Skp KO1 (5’- GTG AAA AAG TGG TTA TTA GCT GCA GGT CTC GGT

TTA GCA Cgt gta ggc tgg agc tgc ttc) and Skp KO2 (5’- TTA TTT AAC CTG TTT CAG

TAC GTC GGC AGT GAT GTC TTT TCA TAT GAa tat gaa tat cct cct tag t). The skp::kan

genotype was verified by PCR using the oligonucleotides Skp1 (5’-ACT CGC GAG CTC

GGG ATG GTA AGG AGT TTA TT) and Skp2 (5’-TCG AAT AAG CTT CAG TCG AAT

TGA AGG CAT TA).

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E. coli UTsurA (UT5600 surA:kan) strain:

The UTsurA strain was obtained by transformation of E. coli UT5600 strain carrying pKD46

with the PCR product obtained after amplification of template pKD4 with the

oligonucleotides SurA KO1 (5’- ATG AAG AAC TGG AAA ACG CTG CTT CTC GGT

ATC GCC ATG Agt gta ggc tgg agc tgc ttc) and SurA KO2 (5’- TTA GTT GCT CAG GAT

TTT AAC GTA GGC GCT GGC ACG TTG TCA TAT GAa tat gaa tat cct cct tag t). The

surA::kan genotype was verified by PCR using the oligonucleotides SurA KO1 and kt. The

sensitiviy of UTsurA strain to SDS 1% in LB-plates was proven.

E. coli UTdegP-PBAD::surA strain:

The zeoRExBAD (zeoR araC PBAD) promoter cassette was amplified from E. coli

TG1zeoRExBAD with oligonucleotides ZEOBAD surA1 (5’- CGC AAG AGA TGC TGC

GTT CGA ACA TTC TGC CGT ATC AAA ACA CTT TGT GAa gca atg ctt gca taa tgt gcc

tgt c) and ZEOBAD surA2 (5’- CTG GTA TTC GCG ATC ATG GCG ATA CCG AGA

AGC AGC GTT TTC CAG TTC TTC CAT cgt ttc act cca tcc aaa aaa acg ggt). The upper-

case letters corresponds to the sequence hybridizing to surA upstream or coding sequences.

The lower case letters corresponds to the sequence hybridizing to the zeoRExBAD cassette.

In bold is the first codon of surA coding sequence. The ~1.9 kb PCR product was

electroporated into E. coli UTdegP carrying pKD46. ZeoR transformant colonies were

selected in low-salt LB containing 0.4% (w/v) arabinose and Zeo (40 µg/ml). The insertion of

the zeoRExBAD cassette in the promoter region of surA was tested by PCR with

oligonucleotides Zeo1 (5’- CAC TGG TCA ACT TGG CCA TGG TTT AG) and SurA2 (5’-

CAT TAA TCC ATC AAC GTC GCT TTC CAG CAC). The synthetic lethality of the

selected transformants upon growth in LB-glucose was confirmed. In the chromosome of

wild-type E. coli, the surA gene is transcribed with downstream genes pdxA, ksgA, apaG and

apaH. These genes are involved in functions unrelated to surA (e.g. pyridoxal 5'-phosphate

biosynthesis and methylation of 16S rRNA). In addition, these genes are transcribed in pdxA-

ksgA-apaG-apaH and apaG-apaH transcripts due to the presence of promoters upstream pdxA

and apaG (http://biocyc.org/ECOLI/).

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E. coli UTPBAD::bamA strain:

The zeoRExBAD (zeoR araC PBAD) promoter cassette was amplified from E. coli

TG1zeoRExBAD with oligonucleotides ZEOBAD bamA1 (5’- AAT GGG GCT TGC ACT

TTT CAA TGA TTT CTC TCG GTT ATG Aag caa tgc ttg cat aat gtg cct gtc) and ZEOBAD

bamA2 (5’- CTA AAC AGC AGC GAC GCT ATG AGC AAC TTT TTC ATC GCC ATc

atc gtt tca ctc cat cca aaa aaa c). The upper-case letters corresponds to the sequence

hybridizing to bamA upstream or coding sequences. The lower case letters corresponds to the

sequence hybridizing to the zeoRExBAD cassette. In bold is the first codon of bamA coding

sequence. The ~1.9 kb PCR product was electroporated into E. coli UT5601 carrying pKD46.

ZeoR transformant colonies were selected in low-salt LB containing 0.4% (w/v) arabinose and

Zeo (40 µg/ml). The insertion of the zeoRExBAD cassette in the promoter region of bamA

was tested by PCR with oligonucleotides Zeo1 (5’- CAC TGG TCA ACT TGG CCA TGG

TTT AG) and bamA2 (5’- GGC CTT CGA AAT GAA TAT CTT TCA C). The lethality of

the selected transformants upon growth in LB-glucose was confirmed. In the chromosome of

wild type E. coli, the bamA gene is transcribed in a single-gene transcript and in a long

transcript with downstream genes skp(hlpA), lpxD, fabZ, lpxAB, rnhB and dnaE. Some of

these genes are involved in OMP, LPS and fatty acid biogenesis (skp, lpxABD, fabZ) but they

are also transcribed in at least two other transcripts skp(hlpA)-lpxD-fabZ-lpxAB-rnhB-dnaE

and lpxD-fabZ-lpxAB-rnhB-dnaE due to the presence of promoters upstream skp(hlpA) and

lpxD (http://biocyc.org/ECOLI/).

E. coli UTdegP-PBAD::bamA strain:

The UTdegP-PBAD::bamA strain was constructed by transformation of UTPBAD::bamA strain

carrying pKD46 with the PCR product obtained after amplification of pKD4 with

oligonucleotides DegP KO3 (5’- CAC AGC AAT TTT GCG TTA TCT GTT AAT CGA

GAC TGA AAT AGT GTA GGC TGG AGC TGC TTC) and DegP KO4 (5’- GAA GAT

GTA TGG AGT TGT GGT GAA GTT CAC AGA TTG TAA GAT ATC CTC CTT AGT).

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The degP::kan genotype was verified by PCR using the oligonucleotides DegP3 (5’- GCA

GAA ACT TTA GTT CGG AAC TTC) and kt (5’- CGG CCA CAG TCG ATG AAT CC).

Plasmid construction

pNeae: a DNA product encoding the IntiminEHEC fragment corresponding to residues 1-659

was obtained by amplification of the chromosome of strain EDL933 with oligonucleotides

Xba1Neae (5’- GCG TAT CTA GAT AAC GAG GGC AAA TCA TGA TTA CTC ATG

GTT GTT ATA CC) and Ba1Neae (5’- GGT ACG GAT CCG GAT ACG GCA CCG GCG

CAC CAT CAA AAA ATA TAA CCG CAC TGG C). This DNA fragment was digested

with XbaI and BamHI and ligated into the purified vector backbone of pHEβ (5), digested

XbaI and HindIII, with the oligonucleotide linker obtained by hybridizing the

oligonucleotides BaEtaghis2 (5'- GAT CCG CTG GAA CCG GCC CAG CCG GCC ATG

GCG CAT CAC CAT CAC CAT CAC GCG GCC GCT TA) and NotEtaghis2 (5'- AGC

TTA AGC GGC CGC GTG ATG GTG ATG GTG ATG CGC CAT GGC CGG CTG GGC

CGG TTC CAG CG).

pInt550: a DNA product encoding the IntiminEHEC fragment corresponding to residues 1-550

was amplified from pNeae plasmid with the oligonucleotides Xba1Neae and IntSfiNco (5’-

GTG ATG CGC CAT GGC CGG CTG GGC CGG TTC CAG CGG ATC CGG ATA CGG

CAC CGG CGC CGA CAG). This amplified DNA product was digested with XbaI and SfiI

and cloned into same sites of the vector backbone pNeae.

pBAD-SurA: The surA gene was amplified with primers SurA-SacI (5’-

ACTCGCGAGCTCGAAATGGAAAAAGTATGAAG) and SurA-XbaI (5’-

TCGAATTCTAGAAACACGTTGGGTTTTAACCA) from the chromosomal DNA of E. coli

K-12 strain DH5α. The resulting ~1.3 kb DNA fagment was digested with XbaI and SacI and

cloned into same sites of pBAD30 (2).

Cell fractionation

A biochemical fractionation was performed to isolate the bacterial OM fraction. Bacteria were

harvested from 200 ml cultures at OD600 ~2. The bacterial pellet was air-dried and frozen at -

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80oC. Stored bacterial pellets were thaw on ice, resuspended in 10 ml of TN buffer (20 mM

Tris-HCl, pH 8.0, 10 mM NaCl) supplemented with DNaseI (Roche) 0.1 mg/ml, pancreatic

RNase A (Amresco) 0.1 mg/ml, PMSF 0.1 mM and a protease inhibitor cocktail (Complete

EDTA-free, Roche). Bacteria were then lysed in a French press (1.000 p.s.i.) and the

recovered protein extracts were centrifuged (1.500 g, 10 min, 4oC) to remove unlysed

bacteria. Cellular envelope and soluble proteins were separated by centrifugation (100.000 g,

1h, 4oC). The resulting pellet was resuspended in 2ml of TN buffer containing 0.15% (v/v)

Triton-X-100 (Sigma) to solubilize the inner membrane proteins. Next, samples were

centrifuged (100.000 g, 1h, 4oC) and the insoluble material corresponds to the OM fraction.

The OMPs in the OM fraction were either extracted with reducing SDS-buffer for standard

SDS-PAGE or solubilized by resuspension in 1 ml of TN buffer containing 1% (w/v)

Zwittergent 3-14 (Calbiochem) for 1h at 4oC for Blue Native PAGE. A final centrifugation

(100.000 g, 1h, 4oC) was performed to recover the solubilized OMPs in the supernatant

(protein concentration ~1 mg/ml).

Blue Native PAGE

BN-PAGE (4) was performed in 4-20% polyacrylamide gradient gels in buffer 50 mM Bis-

Tris-HCl (Fluka), 500 mM 6-aminocaproic acid (Sigma) (pH 7.0), containing 10% (v/v)

glycerol (Merk). Before gel loading, 20 µl of solubilized OMPs (ca. 20 µg) were mixed with

2.5 µl of glycerol 87% (v/v) and 2.5 µl Coomassie Blue G-250 5% (w/v) (Bio-Rad) and kept

on ice. The cathode buffer consisted of 50 mM Tricine (Fluka), 15 mM Bis-Tris-HCl (pH 7.0)

and 0.002% (w/v) Coomasie Blue G250. The anode buffer was 50 mM Bis-Tris-HCl (pH 7.0)

(Fluka). Protein standards of high molecular mass (66-669 kDa) for native electrophoresis

(GE Healthcare) were resuspeded at 2.5 mg/ml final concentration in 50 mM Bis-Tris-HCl

(pH 7.0) containing 750 mM 6-aminocaproic acid. 5-10 µl of OMPs (or resuspended protein

standards) were loaded per well and electrophoresis was run in a Miniprotean III (Bio-Rad)

for 45 min at 100 V and ca. 1 h at 500 V. Proteins were transferred to PVDF membranes and

incubated with antibodies for Western blot as described previously.

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Supplemental References

1. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of

chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640-5.

2. Guzman, L. M., D. Belin, M. J. Carson, and J. Beckwith. 1995. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121-30.

3. Roux, A., C. Beloin, and J. M. Ghigo. 2005. Combined inactivation and expression strategy to study gene function under physiological conditions: application to identification of new Escherichia coli adhesins. J Bacteriol 187:1001-13.

4. Schägger, H., W. A. Cramer, and G. von Jagow. 1994. Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal Biochem 217:220-30.

5. Veiga, E., E. Sugawara, H. Nikaido, V. de Lorenzo, and L. A. Fernández. 2002. Export of autotransported proteins proceeds through an oligomeric ring shaped by C-terminal domains. EMBO J 21:2122-31.

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Supplemental Figures

Figure S1. Mock alkylation of Neae polypeptide (Int550+D0). The Intimin deletion construct Neae (Table 1), lacking Cysteine residues, was

induced in UT5600/pNeae and UTdsbA/pNeae bacteria incubated with DTT and mPEG-MAL as indicated (+ or -) to control the absence of high-

molecular-weight alkylation bands in both strains and to monitor the changes in the electrophoretic mobility of the polypeptide when DTT and/or

mPEG-MAL are present. Samples were subjected to non-reducing SDS-PAGE and Western blot developed with anti-E-tag mAb-POD. The mass of

protein standards is shown on the left (in kDa).

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Figure S2. Phenotypic characterization of E. coli degP, skp and surA null mutants. Western blots probed with anti-Skp (A), anti-DegP (B) or anti-

SurA (C) polyclonal serum of whole-cell protein extracts of E. coli UT5600 and its degP, skp and surA isogenic mutants (Table 1). The asterisks (*)

indicate non-specific cross-reactive protein bands. The anti-DegP polyclonal serum was raised against a fusion of DegP with the maltose binding

protein (MBP) and recognizes MBP and DegP. The mass of protein standards is shown on the left (in kDa).

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Figure S3. Display, folding, outer membrane localization and dimerization of Int550 polypeptide. (A) Flow cytometry analysis to determine that Int550 displays the E-tag epitope on the surface of wild type E. coli UT5600 transformed with pInt550. The same strain was transformed with pNeae (Int550+D0+E-tag) or the empty vector (pAK-Not) as positive and negative controls, respectively. IPTG-induced bacteria were incubated with an anti-E-tag mAb followed by a second incubation with an Alexa 488-conjugated goat anti-mouse IgG antibody and subjected to flow cytometry as described in Materials and Methods. (B) The sensitivity of Int550 to unfold by boiling in SDS or urea-SDS buffers was tested. Whole-cell protein extracts from IPTG-induced E. coli UT5600/pInt550 bacteria were prepared by mixing the bacterial suspension (OD600 of 3.0 in PBS) with the same volume of 2X urea-SDS-sample buffer, or standard SDS-sample buffer, and either boiled or not, as indicated with the (+) and (-) symbols. Western blot developed with anti-E-tag mAb (upper panel) or anti-OmpA (lower panel). The mobility of unfolded (U) and folded (F) polypeptides is labeled on the right. (C) Blue-Native PAGE of solubilized OMPs from the outer membrane factions of E. coli UT5600/pInt550, UT5600/pNeae, and EPEC bacteria. Intimin polypeptides were detected by Western blot incubating the membrane with anti-Int280EPEC serum followed by incubation with a mixture of protein A-POD and anti-E-tag mAb-POD. The mass of native protein standards (GE Amersham) is shown on the left (in kDa).

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Figure S4. Complementation of the sensitivity to SDS of the E. coli surA null mutant by expression of SurA in trans. (A) Growth assay in LB

plate containing 1% SDS and 0.4% L-arabinose of the UTsurA strain transformed with pBAD30 or pBAD-SurA. (B) Western blot probed with anti-

SurA serum of whole-cell protein extracts of UTsurA mutants transformed with pBAD30 or pBAD-SurA grown in LB media containing 0.4% L-

arabinose.

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