Supplemental Table S1. Primers for all the constructs  

Wild

type

NewE-5’ GGGCCATGGgcGTTCGTTGGACCCTGTGGGACACCCTGGCTTTCCTGCTGCTGC

NewE-Rev1 GGGATGAACATGATCAGCAGAGACGGCAGCAGCAGAGACAGCAGCAGCAGGAAAGCCAG

NewE-Fwd2 CTGCTGATCATGTTCATCCCGTCTACCTTCAAACGTCCGGTTTCTTCTTGGAAAGC

NewE-Rev2 CAGAAGAAGCCATCAGCAGGGTTTTACGCAGGTTCAGAGCTTTCCAAGAAGAAACCGG

NewE-Fwd3 CCTGCTGATGGCTTCTTCTGTTCGTCTGAAACCGCTGAACTGCTCTCGTCTGCCGTG

NewE-Rev3 GTCAGCAGGAAGGTCAGGGTTTCCTGAGCGTAAACGCACGGCAGACGAGAGCAG

NewE-Fwd4 CCCTGACCTTCCTGCTGACCCAGAAAAAAACCTGCGTTAAAAACTACGTTCGTAAAG

NewE-3’ GGGGCGGCCGCATTAGTGATGGTGATGGTGATGTTCTTTACGAACGTAGTTTTTAACGC

E-L

S, E

-LN, &

E-L

C

E-LS-5’ GGTGTGCCATGGgtGTTCG

E-LS-Rev1 AGCAGCAGCAGCAGCAGCAGAGTGTCCCACAGGGTCCAACGAACACCCATGGCAC

E-LS-Fwd2 TGCTGCTGCTGCTGCTCTCTCTCCTGCTGCCATCTCTGCTCCTCCTGCTCCTGCC

E-LS-Rev2 GCGCTTTCCAAGAAGAAACCGGACGTTTGAAGGTAGAAGGCAGGAGCAGGAGGAG

E-LS-Fwd3 GGTTTCTTCTTGGAAAGCGCTGAACCTCCGTAAAACGCTGCTCATGGCGAGCTCT

E-LS-Rev3 CACGGCAGACGAGAGCAATTCAGCGGTTTCAGACGAACAGAGCTCGCCATGAGCA

E-LS-Fwd4 TGCTCTCGTCTGCCGTGCGTTTACGCGCAGGAAACCCTGACCTTCCTCCTCACCC

E-LS-Rev4 CTTTACGAACGTAGTTTTTAACGCAGGTTTTTTTCTGGGTGAGGAGGAAGGTCAG

E-LS-Fwd5 GCGTTAAAAACTACGTTCGTAAAGAACACCATCATCACCACCACCTCGAGCACAC

E-LS-3’ GGTGTGCTCGAGGTGGTGG

E-LN-Rev1 TGCTGCTGCTGCTGCTCTCTCTCCTGCTGCCATCTCTGCTCATCATGTTCATCCC

E-LN-Fwd2 GCGCTTTCCAAGAAGAAACCGGACGTTTGAAGGTAGAAGGGATGAACATGATGAGCAG

E-LC-Fwd2 AGCAGCAGCAGGAAAGCCAGAGTGTCCCACAGGGTCCAACGAACACCCATGGCAC

E-LC-Rev2 GGCTTTCCTGCTGCTGCTCTCTCTCCTGCTGCCATCTCTGCTCCTCCTGCTCCTGCC

E25

-28

E-Fwd GGTGTGCCATGGgcGTTCATTGGACCCTGTGGGACAC

E28lyso-Rev GTTCATACCGCTACCGCTGCCGATGAACATGATCAGCAGAGACGGC

E27lyso-Rev GTTCATACCGCTACCGCTGCCGAACATGATCAGCAGAGACGGCAG

E26lyso-Rev GTTCATACCGCTACCGCTGCCCATGATCAGCAGAGACGGCAGAAAC

E25lyso-Rev GTTCATACCGCTACCGCTGCCGATCAGCAGAGACGGCAGAAACAG

Lyso-Fwd GGCAGCGGTAGCGGTATGAACATCTTCGAAATGCTCCGTATCGAC

Lyso-Rev GGTGTGGCGGCCGCATTAGTGATGGTGATGGTGATGCAGGTTTTTGTACGCGTCCCAGG

N-t

erm

inal

de

letio

n

EN9-Fwd GGTGTGCCATGGgaCTGGCTTTCCTGCTGCTGCTGTC

EN10-Fwd GGTGTGCCATGGgaGCTTTCCTGCTGCTGCTGTCTCTG

EN11-Fwd GGTGTGCCATGGgaTTCCTGCTGCTGCTGTCTCTGCTG

EN26-Fwd GGTGTGCCATGGgaTTCATCCCGTCTACCTTCAAACGTCC

E-Rev GGTGTGGCGGCCGCATTAGTGATGGTGATG

*Restriction sites are indicated with underline. Nucleotides added to introduce Gly residue at the position 2 are in lower case.  

Supplemental Figure 1

Fig. S1. Effect of the His6-tag and FLAG-tag on E, alanine-scanning in TOP10, and the effect of various L-rhamnose concentraions in lysis onset. A. Growth curve comparison of wild-type E with His6-tagged or His6-FLAG tagged E. B. Representative growth curves of alanine-scanning mutants in TOP10 cells. Expression of each E construct was induced at time= 0. Western blot of whole cells after 10 min of induction and E in membrane fractions after 60 min of induction. C. Growth curves of wild-type in TOP10 and Lemo21 with different concentrations of L-rhamnose.

WT L15A L18A P21A L18A P21A

B

A

C

0.05

0.1

0.2

0.4

0 10 20 30 40 50 60

A55

0

Time (min)

Control WT L15A L18A P21A

0.05

0.1

0.2

0.4

0.8

0 20 40 60 80 100 120

A55

0

Time (min)

Control TOP10 0mM 0.25mM 1mM 2mM

membrane (60 min)whole cell (10 min)

0.05

0.1

0.2

0.4

0 10 20 30 40 50 60 70 80

A550

Time (min)

Control E (TOP10) E-His6(TOP10) E-His6FLAG(TOP10) E(Lemo21) E-His6(Lemo21) E-His6FLAG(Lemo21)

WT T9 L10 A11 F12 L13 L14 L15 WT V2 R3 W4 T5 L6 W7 D8 WT L16 S17 L18 L19 L20 P21 S22 L23 WT L24 I25 M26 F27 I28 P29

0.05

0.5

0 10 20 30 40 50 60 70 80 90

V2A WT Average

0.05

0.5

0 10 20 30 40 50 60 70 80 90

R3A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

W4A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

T5A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

L6A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

W7A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

D8A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

T9A

0.05 0 10 20 30 40 50 60 70 80 90

L10A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

A11S

0.05

0.5

0 10 20 30 40 50 60 70 80 90

F12A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

L13A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

A55

0

L14A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

L15A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

L16A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

S17A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

L18A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

L19A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

L20A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

P21A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

S22A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

L23A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

L24A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

I25A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

M26A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

Time (min)

F27A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

I28A

0.05

0.5

0 10 20 30 40 50 60 70 80 90

P29A

A

B

Supplemental Figure 2

Fig. S2. Alanine-scanning in Lemo21.A. Raw data of lysis assays of alanine-scanning mutants in Lemo21 cells. At least three replicates growth curves of each mutant were monitored by OD550(grey lines). Average growth curve are shown as solid black lines. For com-parison, the averaged wild-type growth curve is shown as a dotted line. Each mutation is categorized by color as in Fig. 2.B. Western blot of protein levels in whole cells after 20 min of induction.

0 mM L-rhamnose 4mM L-rhamnose

0.05

0.1

0.2

0.4

0.8

0 10 20 30 40 50 60 70 80 90

A55

0

Time (min)

Control WT E-LS E-L E-L

N

C

0.05

0.1

0.2

0.4

0.8

0 20 40 60 80 100 120

A55

0

Time (min)

Control WT E-LS E-L E-L

N

C

Supplemental Figure 3

Fig. S3. Growth curves of leucine mutants in Lemo21. Cultures grown with 0 mM L-rhamnose (left) or 2 mM L-rhamnose (right).

0.1

0.2

0.4

0.8

0 10 20 30 40 50 60

Control

WT

E-MBP

E-Lysozyme

Supplemental Figure 4

Fig. S4. A. Growth curves of E-MBP and E-lysozyme fusion constructs.B. Growth curve of E28 and E11-28(L19F) in the SlyD knockout strain.

B

A

A55

0 A

550

SlyD knockout strain

Time (min)

Time (min)

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 10 20 30 40 50 60 70 80 90

Control WT E28

E11-28(L19F)

Supplemental Table S2. Predicted apparent free energy difference for insertion of the Phe mutants

 Mutants Sequence of TM Predicted ΔG E11-28 MGAFLLLLSLLLPSLLIMFI -1.470 E11-28(L13F) MGAFFLLLSLLLPSLLIMFI -1.351 E11-28(L14F) MGAFLFLLSLLLPSLLIMFI -1.303 E11-28(L15F) MGAFLLFLSLLLPSLLIMFI -1.343 E11-28(L16F) MGAFLLLFSLLLPSLLIMFI -1.368 E11-28(L18F) MGAFLLLLSFLLPSLLIMFI -1.297 E11-28(L19F) MGAFLLLLSLFLPSLLIMFI -1.359 E11-28(L19I) MGAFLLLLSLILPSLLIMFI -1.502 E11-28(L20F) MGAFLLLLSLLFPSLLIMFI -1.347 E11-28(L23F) MGAFLLLLSLLLPSFLIMFI -1.378 E11-28(L24F) MGAFLLLLSLLLPSLFIMFI -1.338 * Values for predicted ΔG were calculated using the prediction of ΔG for TM helix insertion website at http://dgpred.cbr.su.se/index.php?p=TMpred.