Supporting Online Material for “Contact-dependent Inhibition of Growth in Escherichia

coli.”

Stephanie K. Aoki, Rupinderjit Pamma, Aaron D. Hernday, Jessica E. Bickham, Bruce A. Braaten, and David A. Low

MATERIALS AND METHODS

Strains, plasmids, and growth conditions

E. coli strains and plasmids used in this study are listed in table S1. Cells were grown on

Luria-Bertani (LB) broth or agar supplemented with antibiotics at the following

concentrations (unless otherwise noted): ampicillin (amp), 100 µg/ml; chloramphenicol

(cam), 34 µg/ml; kanamycin (kan), 40 µg/ml; nalidixic acid (nal), 10 µg/ml; streptomycin

(str), 100 µg/ml; tetracycline (tet), 12.5 µg/ml; rifampicin (rif), 150 µg/ml. Cultures were

incubated at 37°C in an environmental shaker apparatus (New Brunswick Series 25) at

225 rpm unless otherwise indicated.

Competition assay

E. coli EC93 and EPI100 CDI+ (DL4577) inhibitory cells were separately grown to

logarithmic phase (OD600 ≈ 0.35) in 50 ml LB medium containing appropriate antibiotics

(500 ml baffled culture flask). EPI100 CDI– (DL4527) cells were also included as a no

inhibition control. MG1655 (DL4793) target cell cultures (16 h) were added at a 10 to1

inhibitor to target cell ratio. Competitions were incubated at 37°C with shaking at 225

rpm. Samples were taken hourly, ten-fold serially diluted in M9 salt solution (Sigma

Co.) and 100 µl aliquots of serial dilutions were plated onto LB containing appropriate

antibiotics for independent selection of inhibitor and target cells.

1

Cosmid library construction and nested deletions

Cosmid library construction (pDAL660)

A cosmid library of E. coli EC93 genomic DNA was made with an Epicentre

pWEB::TNC Cosmid Cloning Kit. Briefly, DNA (~ 40 kb) was blunt-end cloned into the

SmaI site of pWEB::TNC, packaged using MaxPlax Lambda Packaging Extracts and

transduced into EPI100 cells. Transductants were selected on LB-amp-cam agar and

screened for growth inhibitory activity against MC4100 ampR kanR (DL159) target cells

by incubating transductant and target cells together in LB containing appropriate

antibiotics for 3 hours before sampling. Samples were ten-fold serially diluted in M9 salt

solution and 5 µl aliquots were spotted onto LB-kan. CDI activity was scored by

observation of the spotted target cell growth (single colonies versus confluent growth).

Nested deletions (pDAL660∆)

Nested deletions within pDAL660 were constructed with an Epicentre pWEB::TNC

Deletion Cosmid Transposition Kit. Briefly, EZ::TN Transposase was used in vitro to

generate unidirectional deletions, designated as pDAL660∆X where “X” specifies a

particular deletion. Deletion plasmids were transformed into EPI100 and transformants

were selected for ampicillin-resistance and chloramphenicol-sensitivity. The positions of

deletions were determined by sequencing using the pWEB::TNC sequencing primer

(table S1). Deletion mutants were screened for growth inhibitory activity against EPI100

target cells (DL4527) by incubating transformant and target cells together in LB

containing appropriate antibiotics for 3 hours before sampling. Samples were ten-fold

serially diluted in M9 salt solution and 5 µl aliquots were spotted onto LB-cam. CDI

2

activity was scored by observation of the spotted target cell growth (single colonies

versus confluent growth).

Linker insertions

TnsABC* transposase (New England Biolabs) was used to randomly introduce single

hops of a Tn7-based transprimer into deletion subclone pDAL660∆1-39. Transposon

insertion sites were determined by DNA sequence analysis using oligonucleotide primers

N and S (table S1) (see GPS-LS Linker-Scanning System, New England Biolabs). To

convert transposons to 15bp linkers, each plasmid containing a transprimer insertion was

digested with PmeI, ligated, and transformed into E. coli EPI100. This resulted in

replacement of the transprimer sequence of each plasmid with a 15 bp insertion that

either introduced an in-frame five amino acid sequence or an in-frame stop codon,

designated as pDAL660∆1-39::TP–. Transformants were screened for growth inhibitory

activity against MG1655 (DL4793) target cells by incubating transformant and target

cells together in LB containing appropriate antibiotics for 3 hours before sampling.

Samples were ten-fold serially diluted in M9 salt solution and 5 µl aliquots were spotted

onto LB-tet. CDI activity was scored by observation of the spotted target cell growth

(single colonies versus confluent growth).

FLAG-tag Linker Insertion (pDAL660∆1-39::FLAG)

Plasmid pDAL660∆1-39::TP¯containing five amino acid insertions within CdiA (mutant

numbers 1-64, 1-14, 1-67, 1-39, and 2-17) and CdiB (1-33) (see Fig. 2B and table S2)

were linearized by digesting with PmeI. The appropriate FLAG-tag linker DNA

sequence encoding DYKDDDDK (table S1, oligonucleotide 865) was blunt-end cloned

3

in-frame into the PmeI site and electroporated into TransforMax EPI100 (Epicentre).

AmpR colonies were screened for correct FLAG orientation by PCR, and assayed for

growth inhibitory activity and immunity as described above.

Screens and quantitative assays for growth inhibition and immunity

A screen to identify bacteria with contact-dependent growth inhibitory activity (CDI) was

developed to test cosmids, cosmid subclones, and transposon insertions as follows.

Bacteria to be screened were grown to logarithmic phase (OD600 ≈ 0.8) in 5 ml LB

medium containing appropriate antibiotics. Overnight cultures (16 h) of target cells were

added for an inhibitor to target cell ratio of 20 to 1. Competitions were then incubated for

3 h, ten-fold serially diluted in M9 salt solution, and aliquots (5 µl) of each dilution were

spotted onto LB agar containing appropriate antibiotics for selection of target cells. CDI

activity was scored by observation of the spotted target cell growth (single colonies

versus confluent growth). EC93 or DL4577 was used as a positive control for inhibitory

activity and EPI100 was used as a negative control for inhibitory activity in each

experiment. A quantitative assessment of growth inhibition was carried out as above

except that 100 µl aliquots of serial dilutions were plated on LB containing appropriate

antibiotics to obtain viable colony counts of target cells.

A screen to detect bacteria immune to CDI-dependent growth inhibition was carried out

by inoculating overnight cultures (16 h) of strains to be screened into LB cultures of

logarithmic phase DH5α CDI+ (DL4608) inhibitory cells for an inhibitor to target ratio of

20 to 1. After 3 h of incubation, cultures were ten-fold serially diluted and spotted as

above for the growth inhibition assay onto LB-str agar for selection of strains being

tested. E. coli EC93 strR was used as a positive control and EPI100 was used as a

4

negative control for immunity to CDI. Quantitation of immunity was done as above for

immunity screening except that 100 µl aliquots were plated on LB-str agar for viable

colony counts of the tested strains.

Growth phase assay

E. coli CDI+ EC93 inhibitor cells and MC4100 NalR (DL430) target cells were separately

incubated in LB medium and grown to stationary phase (16 h) or logarithmic phase

(OD600 ≈ 0.35). Inhibitor cells were then mixed with target cells and incubated at 37°C

with shaking (225 rpm). Stationary phase inhibitor cells were incubated with target cells

at an inhibitor to target cell ratio of 500 to 1. Logarithmic phase inhibitor cells were

incubated with target cells at an inhibitor to target cell ratio of 5 to 1. Samples were

taken hourly, ten-fold serially diluted in M9 salt solution and 100 µl aliquots were plated

onto LB containing appropriate antibiotics for independent selection of inhibitor and

target cells. E. coli MC4100 (K-12) CDI– inhibitor control cells were also included as a

negative control for growth inhibitory activity.

Chloramphenicol pretreatment assay

E. coli EPI100 CDI+ inhibitor (DL4577) and EPI100 CDI– inhibitor control (DL4956)

cells were separately inoculated into 50 ml of LB-amp medium in a 500 ml baffled

culture flask and grown to logarithmic phase (OD600 ≈ 0.3). Chloramphenicol was then

added to a final concentration of 34 µg/ml and cultures were incubated for two additional

hours. Target E. coli DL4793 culture (16 h) was added to chloramphenicol pretreated

cells at an inhibitor to target ratio of 10 to 1. Samples were taken hourly, ten-fold serially

diluted in M9 salt solution and 100 µl aliquots were plated onto LB containing

appropriate antibiotics to obtain viable colony counts of inhibitor and target cells.

5

Untreated inhibitor and inhibitor control cultures were included as a negative control for

protein synthesis inhibition.

Analysis of CDI at different inhibitor to target cell ratios

Logarithmic growth phase E. coli DL4577 (OD600 ≈ 0.35, 15 mL) were added to 125 mL

flasks and inoculated with target E. coli DL4793 at different inhibitor to target cell ratios.

Cells were incubated at 37°C with shaking (150 rpm) and samples were obtained at 0, 5,

20, 60, 75, 90, 120, 135, 150, and 180 minutes. Samples were ten-fold serially diluted in

M9 salt solution and plated on LB containing appropriate antibiotics for independent

selection of inhibitor and target cells. CDI- E. coli DL4527 was used as a negative

control for growth inhibitory activity.

Growth Inhibitory Activity Secretion Assays

E. coli EPI100 CDI+ inhibitor (DL4577) and EPI100 CDI– control cells were separately

grown to logarithmic phase (OD600 ≈ 0.35) in LB-amp medium and 16 h target MG1655

(DL4793) cells were added at a 10 to1 inhibitor to target cell ratio. Samples were taken

hourly, ten-fold serially diluted in M9 salt solution and 100 µl aliquots were plated onto

LB containing appropriate antibiotics to obtain viable counts of inhibitor and target cells.

After one hour of co-incubation, a 15 ml sample from each culture was centrifuged at

6000 x g for 1 min and supernatant solutions were filtered through a low protein binding

0.45 µm HT Tuffryn membrane (Pall Life Sciences) to remove any remaining cells.

Target E. coli DL4793 cells (16 h culture) were added 1/200 (v/v) into 12.5 ml of filtered

cell culture supernatant. Samples were taken hourly and viable target cell counts were

determined as described above.

Contact-dependent inhibitory activity assay

6

Polyethylene terephthalate (PET) track-etched membrane inserts (23 mm) of 0.4 µm or 8

µm pore size (Falcon) were placed in 6-well plates to create upper and lower culture

wells. LB-amp medium (15 ml in a 125 ml flask) was inoculated with an overnight

inhibitor culture of EPI100 CDI+ (DL4577), grown to logarithmic phase (OD600 ≈ 0.35),

and 2.5 ml was added to the top chambers. 3.2 ml of a 16 h culture of target MG1655

(DL4793) cells was added to the bottom chambers at a 20 to 1 ratio of inhibitor to target

cells (estimated by OD600). Cells were incubated at 37°C with shaking (130 rpm) and

sampled at 0, 1, 3, and 5 hours. Samples were ten-fold serially diluted in M9 salt solution

and 100 µl aliquots were plated onto LB containing appropriate antibiotics for

independent selection of inhibitor and target cells. E. coli EPI100 CDI– (DL4527) was

used in place of DL4577 as a negative control for inhibitory activity.

PET membrane control

Several PET membranes (Falcon) were minced aseptically with dissecting scissors. PET

membrane fragments (1.5 membranes/well) together with logarithmic phase CDI+

inhibitory strain DL4577 were added to the top chamber of 6-well plates containing a 0.4

µm membrane insert. Membranes were incubated with inhibitor cells for 5 minutes at

37°C with shaking before adding DL4793 target cells (16 hour culture) at a 20 to1 ratio

of inhibitor to target (estimated by OD600). Cells were incubated at 37°C with shaking

(130 rpm) and samples were obtained at 0, 1, 3, and 5 hours. Samples were ten-fold

serially diluted in M9 salt solution and plated onto LB containing appropriate antibiotics

for independent selection of inhibitor and target cells. Wells containing DL4577 and

DL4793 without membrane fragments were used as a positive control of target cell

inhibition.

7

FACS analysis

Flow cytometry was carried out using a BD Biosciences FACSAria instrument with a

100µm sorting nozzle at low pressure. GFP-mut3 and DsRed were excited using a

488nm blue laser and detected using 530/30nm and 610/20nm filters respectively.

DsRed labeled target E. coli were prepared by overnight growth of DL4920 in TB-amp

broth with 0.8mM IPTG to induce expression of DsRed from pDAL672. Overnight

cultures of both target and inhibitor E. coli were separately diluted 100-fold into fresh

TB-amp broth with 0.8mM IPTG and grown to early log phase (OD600 = 0.2) prior to

mixing. Mixed cultures (6ml) were incubated at 37ºC in 50 ml baffled culture flasks

with shaking at 250 rpm for the times indicated (Fig. 2, D and E). Inhibitor-bound target

cells and free target cells were isolated by sorting 10,000 particles of each cell population

into separate tubes containing phosphate buffered saline (1 ml). Samples were then

vortexed vigorously (45s) to disrupt intercellular aggregates and plated in quadruplicate

(0.1ml per plate) on LB-tet (5 µg/ml) agar plates. Target cell viability was determined by

counting colony-forming units per particle sorted from each gated population.

Immunoblot analysis

EPI100 pDAL660∆1-39::FLAG strains and DL4577 (no FLAG, negative control) were

grown in LB medium to an OD600 = 0.8, and 150 µl of culture was centrifuged at 16,000

x g for 30 s. Pellets were resuspended in 30 µl LDS sample buffer with reducing agent

(Invitrogen). Samples were incubated at 75°C for 15 minutes and loaded onto a 12-well

NuPAGE Novex 3-8% Tris-acetate gel (Invitrogen) along with 1 µl Odyssey protein

molecular weight marker (LI-COR) and electrophoresis was performed according to

manufacturer (Invitrogen). Proteins were transferred to nitrocellulose in Tris-glycine

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transfer buffer using the XCell II Blot Module (Invitrogen) as directed by manufacturer.

The membrane was rinsed with phosphate-buffered saline (PBS) and blocked overnight

at 4°C in Odyssey blocking buffer:PBS (1:1). Mouse anti-FLAG M2 antibody (Sigma)

1:500 in Odyssey blocking buffer:PBS was added to the membrane and incubated for 1 h

at room temperature. Blots were rinsed in PBS + 0.05% Tween-20 prior to incubation

with goat anti-mouse IgG Alexa Fluor 680-allophycocyanin (Molecular Probes) 1:2500

in Odyssey blocking buffer:PBS + 0.1% Tween-20 for 1 h at 23˚C. The blot was washed

in PBS + 0.1% Tween-20, then PBS, and air-dried overnight. Blots were imaged on the

700 channel of the Odyssey Infrared Imaging System (LI-COR).

Complementation analysis using cdiA and cdiB genes from UPEC strains

cdiB complementation

Homologues of cdiB from uropathogens A8, A50, J96, 3576H, and 536 were amplified

by polymerase chain reaction (PCR) using oligonucleotide primers 854 and 855 (table

S1) based on the cdiB DNA sequence from EC93. The 1.8 kb PCR products were blunt-

end cloned into the Eco72I site of plasmid vector pCC1 under lac promoter control using

the Copy-Control PCR Cloning Kit (Epicentre) according to manufacturer instructions.

Briefly, PCR products were precipitated, end-filled and ligated into the Eco72I site of

pCC1. Ligations were electroporated into TransforMax EPI300 (Epicentre) and

chloramphenicol-resistant colonies were screened for insert orientation.

Complementation was carried out by transforming cloned cdiB genes into E. coli EPI300

pDAL660∆1-39::2-7TP– (DL4958), which contains a non-polar 5 amino acid insertion

within the cdiB gene and lacks growth inhibitory activity but maintains immunity (table

S2). DL4958 transformed with EC93 cdiB (pDAL662) was used as a positive control for

9

complementation and DL4958 containing plasmid pCC1 was used as a negative control

for complementation. Strains to be tested were grown to logarithmic phase (OD600 ≈

0.35) in 12.5 ml LB containing appropriate antibiotics in the presence of CopyControl

induction solution (Epicentre) for maximal CdiB expression. 16 h target cell cultures

(MG1655, DL4793) were added at an inhibitor to target cell ratio of 10 to1.

Competitions were incubated at 37°C with shaking at 225 rpm and sampled hourly.

Samples were ten-fold serially diluted in M9 salt solution and 100 µl aliquots were plated

onto LB containing appropriate antibiotics for independent selection of inhibitor and

target cells.

cdiA complementation

For complementation with cdiA, an 11 kb DNA fragment from UPEC 536 encompassing

cdiA was amplified using PCR and oligonucleotide primers 875 and 877 (table S1). The

fragment was blunt-end cloned into the Eco72I site of pCC1 as for cdiB above to

generate plasmid pDAL665. Complementation was carried out by transforming the

plasmid into E. coli EPI300 containing pDAL660∆1-39::1-32TP– (DL4957), which

contains a stop codon within the cdiA gene and lacks growth inhibitory activity but

maintains immunity (table S2). Complementation was tested as for cdiB above.

Cloning and analysis of cdiI

A 401 bp DNA fragment adjacent to cdiA (Fig. 3A , bp 11,063 to 11,464) was amplified

from E. coli EC93 by PCR using oligonucleotides 883 and 884 (table S1). The DNA

fragment was blunt-end cloned into vector pCC1 as described earlier, electroporated into

EPI300, and chloramphenicol-resistant colonies were screened for insert orientation.

Plasmids pDAL664 and pDAL664-REV were generated containing a lac promoter

10

controlling the putative ORF designated cdiI in the same transcriptional orientation or

reverse orientation, respectively. Analysis of immunity was performed by growing CDI+

inhibitor cells (DL4577) to logarithmic phase (OD600 ≈ 0.35) and adding transformant

target cells [16 h growth in LB-cam medium with CopyControl induction solution

(Epicentre) to maximize cdiI expression] at an inhibitor to target ratio of 20 to 1. Mixed

cultures were incubated at 37ºC with shaking (225 rpm) and sampled hourly. Samples

were ten-fold serially diluted in M9 salt solution and plated on LB containing appropriate

antibiotics for viable counts of inhibitor and target cells.

Pili expression and resistance to inhibition

E. coli HB101 rifR target cells (pili-) were transformed with plasmids expressing P pili

(pDAL230B), S pili (pANN801-13), type 1 pili (pSH2), or plasmid vector alone

(pBR325)(table S1). Target cells (16 h) were incubated with logarithmic phase (OD600 ≈

0.35) CDI+ inhibitory E. coli (DL4577), and samples were taken three hours after

incubation, serially diluted in M9 salt solution and plated onto LB containing appropriate

antibiotics for viable counts of inhibitor or target cells. No inhibition of target cell

growth was observed using CDI- E coli DL4527 in place of CDI+ DL4577 inhibitory

cells, showing that the growth inhibition observed in Fig. 4 is CDI-dependent (S13).

11

FIGURES

Fig. S1.

0 2 4 6102

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106

108

1010

Time (h)

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et c

ells

(cfu

/ml)

0 2 4 6102

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1010

Time (h)

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(cfu

/ml)

0 2 4 6102

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1010

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/ml)

0 2 4 6102

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1010

Time (h)

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et c

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(cfu

/ml)

A B

C D

Fig. S1. Dependence of CDI on cellular growth phase. E. coli CDI+ EC93 inhibitor cells and E. coli MC4100 (K-12) CDI– control cells were separately grown in LB medium to stationary phase (16 h) or logarithmic phase (OD600 ≈ 0.35). EC93 and MC4100 cells were then mixed with E. coli MC4100 nalR target cells and incubated with shaking (225 rpm) for various times. The viability of target E. coli grown in the presence of EC93 inhibitory cells (red squares) or CDI- MC4100 control cells (black triangles) is plotted on the y-axis. (A) Stationary phase inhibitor or control cells incubated with stationary phase target cells (inhibitor or control to target cell ratio of 500/1) (B) Stationary phase inhibitor or control cells incubated with logarithmic phase target cells (inhibitor or control to target cell ratio of 500/1) (C) Logarithmic phase inhibitor or control cells incubated with stationary phase target cells (inhibitor or control to target cell ratio of 5/1) (D) Logarithmic phase inhibitor or control cells incubated with logarithmic phase target cells (inhibitor or control to target cell ratio of 5/1). Fig. S2.

0 2 4 6101

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109

Time (h)

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et c

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(cfu

/ml)

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Fig. S2. Chloramphenicol pretreatment and inhibition recovery assay. E. coli EPI100 CDI+ (DL4577) and EPI100 CDI– (DL4527) cells were separately grown to logarithmic phase in LB-amp medium (OD600 ≈ 0.35). Cultures were pretreated with chloramphenicol (34 µg/ml) for 2 h before adding E. coli DL4793 target cells (16 h) at a 10/1 inhibitor or control to target ratio. An untreated (no cam) competition was included as a positive control for the inhibition of target cells. Competitions were incubated at 37°C with shaking (225 rpm) and viable target cell counts were determined at the times indicated. Target cells incubated with CDI+ inhibitory cells are shown by triangles and target cells incubated with CDI– control cells are shown by squares. Cam pretreatment is represented by open symbols and the no treatment control is represented by red symbols. Fig. S3.

0 30 60 90 120 150 18010-2

100

102

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1:101:110:1

1:101:110:1

Initial CDI+Inhibitor to Target Ratio: Initial CDI- Control to Target Ratio:

Time (min)

Inhi

bito

r to

targ

et ra

tio

Fig. S3. Analysis of CDI under different inhibitor to target cell ratios. E. coli CDI+ inhibitor (DL4577) cells were grown to logarithmic phase (OD600 ≈ 0.35). Inhibitor cells (15 mL) were added to flasks (125 ml) and inoculated with DL4793 target cells (16 h culture) at the inhibitor to target cell ratios indicated. Mixed cultures were incubated at 37°C with shaking (150 rpm) and viable counts of inhibitory and target cells were obtained and plotted as a ratio at the times indicated. CDI– strain DL4527 was used in place of DL4577 as a negative control for inhibitory activity. 13

Fig. S4.

CDI+ E. coli e growth medium. (A) E. coli EPI100

d at a

h

Fig. S5.

Fig. S5. PET membrane control. PET membranes were minced aseptically with dissecting scissors and added

0 2 4 6101

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Supernatant CollectedTime (h)

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0 2 4 6107

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A

B

Fig. S4. do not release growth inhibitory activity into thCDI+ (DL4577) inhibitor and EPI100 CDI– (DL4527) inhibitor control cells were separately grown to logarithmic phase (OD600 ≈ 0.35) in LB-amp medium. Target MG1655 tetR (DL4793) cells were adde10/1 inhibitor or control cell to target cell ratio. Viable DL4793 target cell counts obtained after addition of CDI+ inhibitor (red squares) or CDI- inhibitor control (black triangles) cells for the times indicated are shown. (B) At the one hour time point in panel A above (indicated by arrow), a 15 ml sample from eacculture was centrifuged at 6000 x g for 1 min and supernatant solutions were filtered through a low proteinbinding 0.45 µm HT Tuffryn membrane (Pall Life Sciences) to remove any remaining cells. A stationary phase culture (16 h) of target E. coli DL4793 was added 1/200 (v/v) into 12.5 ml of filtered cell culture supernatant, and viable target cell counts were determined at the times indicated. Results obtained after addition of filtered supernatants of CDI+ mixed cultures (red squares) or CDI- mixed cultures (black triangles) are shown.

0 1 3 5101

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Time (h)

Tota

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to

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. Analysis of the immunity region linked to cdiAB. (A) The cdiI ORF was cloned in opposite orientations in plasmid pCC1 to generate plasmi

cdiI and plasmid pDAL664-REV in the reveed into cdiABI - E. coli K-12 strain EPI300, and tested for immunity to CDI

scribed in Materials and Methodsack squares, EPI300(pDAL664-REV)

EPI300 containing plasmid pCC1 alone is also shown (red triangles).

0 1 2 3 4 5100

102

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106

Time (h)

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et c

ells

(cfu

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top wells of a 6-well plate (Falcon, 1.5 membranes per well) together with logarithmic phase CDI+ DL4577. in of incubation at 37°C with shaking, DL4793 target cells (16 h culture) were added at a 20/1

inhibitor to target ratio. Mixed cultures were incubated at 37°C with shaking (130 rpm) and viable counts were determined at the times indicated (filled bars). A control lacking added PET membrane is shown by open bars.

Fig. S6.

Fig. S6 from E. coli EC93 (Fig. 3A) d pDAL664 with the lac promoter in

the same orientation as rse orientation. Plasmids were individually transform + inhibitory cells (DL4577) as de . (B) Results obtained using EPI300(pDAL664) are depicted by bl are depicted by black circles and control E. coli

After 5 m

11,063 11,464

lacp

cdiI11,464

lacp

pDAL664-REV

pDAL664

108

A

B

cdiI

Fig. S7. 1 50 CdiA_K93 (1) -------------------------------------------MHQPPVR CdiA_EC 536 (1) -------------------------------------------MHQPPVR CdiA_B_pseudo (1) MNKNRYRVVFNRARGALMVVQENGRASHG--SGSRDASAGVVPAWLSLSP CdiA_Y_pestis (1) MNKNLYRIVFNQARGMLMVVADIAASGRAASSPSSGV

16GAGVVSAGKLASGGGAVNV

GHTQRRRVSALSP CdiA_pertussis (1) MNTNLYRLVFSHVRGMLVPVSEHCTVGNT--FCGRTRGQARSGARATSLS 51 100 CdiA_K93 (8) FTYRLLSYLVSAIIAGQPLLPAVGAVITPQNGAGMDKAANGVPVVNIATP CdiA_EC 536 (8) FTYRLLSYLISTIIAGQPLLPAVGAVITPQNGAGMDKAANGVPVVNIATP CdiA_B_pseudo (49) FALRHVALAVLVAAGVVPIWVNAQVVAGGAHAPSVIQTQNGLQQVNINRP CdiA_Y_pestis (51) LSFRLLIALGCISLSAQAAIVADGSAPGNQQ-PTIISSANGTPQVNIQTP CdiA_pertussis (49) VAPNALAWALMLACTGLPLVTHAQGLVPQGQ-TQVLQGGNKVPVVNIADP 101 150 CdiA_K93 (58) NGAGISHNRFTDYNVGKEGLILNNATGKLNPTQLGGLIQNNPNLKAGGEA CdiA_EC 536 (58) NGAGISHNRFTDYNVGKEGLILNNATGKLNPTQLGGLIQNNPNLKAGGEA CdiA_B_pseudo (99) GASGVSMNTYNQFDVPKPGIILNNSP-INVQTQLGGIIGGNPNFQAGDAA CdiA_Y_pestis (100) SSGGVSRNAYRQFDVDNRGVILNNGRG-VNQTQIAGLVDGNPWLARG-EA CdiA_pertussis (98) NSGGVSHNKFQQFNVANPGVVFNNGLT-DGVSRIGGALTKNPNLTRQ--A 151 200 CdiA_K93 (108) KGIINEVTGGKRSLLQGYTEVAGKAANVMVANPYGITCDGCGFINTPHAT CdiA_EC 536 (108) KGIINEVTGGNRSLLQGYTEVAGKAANVMVANPYGITCDGCGFINTPHAT CdiA_B_pseudo (148) RLIVNQVNSNNPSFIRGKVEIGGAAAQLVIANQAGLVVDGGGFLNTSRAT CdiA_Y_pestis (148) SVILNEVNSRDPSQLNGYIEVAGRKAQVVIANPAGITCEGCGFINANRAT CdiA_pertussis (145) SAILAEVTDTSPSRLAGTLEVYGKGADLIIANPNGISVNGLSTLNASNLT 201 250 CdiA_K93 (158) LTTGKPVMNADGSLQALEVTEGSITINGAGLDGTRSDAVSIIARATEVNA CdiA_EC 536 (158) LTTGRPVMNADGSLQALEVTEGSITINGAGLDGTRSDAVSIIARATEVNA CdiA_B_pseudo (198) LTTGNPNFGPDGSLTGFNVNQGLISVVGAGLDTANVDQVDLLARAVQINA CdiA_Y_pestis (198) LTTGQAQLN-NGQLTGYDVERGEIVIQGKGLDSRGQDHTDLIARSVKVNA CdiA_pertussis (195) LTTGRPSVN--GGRIGLDVQQGTVTIERGGVNATGLGYFDVVARLVKLQG 251 300 CdiA_K93 (208) ALHAK------DLTVTAGANRITADGRVSALKGEG----NVPKVAVDTGA CdiA_EC 536 (208) ALHAK------DLTVTAGANRITADGRVSALKGEG----DVPKVAVDTGA CdiA_B_pseudo (248) KAYAK------TLNVVAGSNQVDYNTLNATPIAANG---PAPTIAIDVSQ CdiA_Y_pestis (247) GIWAN------ELNITTGRNQVDAAHQNINTNAADGR--HRPAVAVDVAN CdiA_pertussis (243) AVSSKQGKPLADIAVVAGANRYDHATRRATPIAAGARGAAAGAYAIDGTA 301 350 CdiA_K93 (248) LGGMYARRIHLTSTESGVGVN-LGNLYAREGDIILSSSGKLVLKNSLAGG CdiA_EC 536 (248) LGGMYARRIHLTSTESGVGVN-LGNLYARDGDIILSSAGKLVLKNSLAGG CdiA_B_pseudo (289) LGGMYANRVFLVSSENGVGVANAGDIAAQAGDLTLQANGRLVLSGHTNAA CdiA_Y_pestis (289) LGGMYAGKIRLIGTETGVGVHNAGEIGASAGDIVITADGMLVNRGQISSA CdiA_pertussis (293) AGAMYGKHITLVSSDSGLGVRQLGSLSSPSAITVSSQGEIALGDATVQRG 351 400 CdiA_K93 (297) NTTVTGTDVSLSGDNKAGGNLSVTGTTGLTLNQSRLVTDKNLV------- CdiA_EC 536 (297) NTTVTGTDVSLSGDNKAGGNLSVTGTTGLTLNQPRLVTDKNLV------- CdiA_B_pseudo (339) GNMSLSASGGIQNSGVTYGKQSVTITTGTDLTNSG--------------- CdiA_Y_pestis (339) QQLAVNTPSGIENSGVLYGKGNTQLTTAGKLSNSG-------T-------

CdiA_pertussis (343) PLSLK AGGGAVKIASASSVGNLAVQGGGKVQ

401 450 CdiA_K93 (340) --LSSSGQIVQNGGELTAGQNAMLSAQHLNQTSGTVNAAENVTLTTTDDT CdiA_EC 536 (340) --LSSSGQIVQNGGELTAGQNAMLSAQHLNQTSGTVNAAENVTLTTTNDT CdiA_B_pseudo (374) ---ALTAQQNLTANVGSLNSTGTLGAGINVDSTVSTSGDLNVTSSGQLTA CdiA_Y_pestis (375) --VAAAGDTLIRAAEVNSSRNSVLGAGIKSDNSAITRGTLDIKARGQLTA CdiA_pertussis (393) ATLLNAGGTLLVSGRQAVQLGAASSRQALSVNAGGALKADKLSATRRVDV 451 500 CdiA_K93 (388) TLKGRSVAGKTLTVSSGSLNNGGTLVAGRDATVKTGTFSNTGTVQGNGLK CdiA_EC 536 (388) TLKGRSIAGKTLTVSSGSLNNGGTLVAGRDATVKTGTFSNTGTVQGNGLK CdiA_B_pseudo (421) TGTNSAAGNATFTGSGINLSNSATAANGNLALSATAGDVNLAGSTVSAKG CdiA_Y_pestis (423) QGKNISGTAQTFNANRIDLSGSQTQSGDLTFTTEGGDIDLTGANLFANRR CdiA_pertussis (443) DGKQAVALGSASSNALSVRAGGALKAGKLSATGRLDVDGKQAVTLGSVAS 501 550 CdiA_K93 (438) VTATDLTSTGSIKSGSTLDISARNA----------TLSGDAGAKDRALVT CdiA_EC 536 (438) VTATDLTSTGSIKSGSTLDISARNA----------TLSGDAGAKDSARVT CdiA_B_pseudo (471) AVNAQASGTVINDRGNLSSGAGMTL----------GGGSLSNQGGRANSQ CdiA_Y_pestis (473) LSVSTPSLLRTDKANLFAEQIALDA----------QALANVGGVITQTGL CdiA_pertussis (493) DGALSVSAGGNLRANELVSSAQLEVRGQREVALDDASSARGMTVVAAGAL 551 600 CdiA_K93 (478) VSGTLENRGRLVSDDVLTLSATQINNSGTLSGAKELVASADTLTTTEKSV CdiA_EC 536 (478) VSGTLENRGRLVSDDVLTLSATQINNSGTLSGAKELVASADTLTTTEKSV CdiA_B_pseudo (511) GPLSVQMAGTVSNQNGMLSSQSTADVRGSAIQNNAGLIQSAGKQTIAGAS CdiA_Y_pestis (513) TDFNLNLPGDIDNRDGTLLTRGNFLLQAEHLTSNSQSLLGAGIQSDGKLA CdiA_pertussis (543) AARNLQSKGAIGVQGGEAVSVANANSDAELRVRGRGQVDLHDLSAARGAD 601 650 CdiA_K93 (528) TNSDGNLMLDSASSTLAGETSAGGTVSVKGNSLKTTTTAQTQGNSVSVDV CdiA_EC 536 (528) TNSDGNLMLDSASSTLAGETSAGGTVSVKGNSLKTTTTAQTQGNSVSVDV CdiA_B_pseudo (561) IDNSAGRLISLNADGLSVTATGALTNAAGANVSGDPGGVIGGKGDVTVQG CdiA_Y_pestis (563) PRGDLNVTTRHALIAQGKTLTAGTLALSGSRLDLTDSLTQAKYMRLTATE CdiA_pertussis (593) ISGEGRVNIGRARSDSDVKVSAHGALSIDSMTALGAIGVQAGGSVSAKDM 651 700 CdiA_K93 (578) QNAQLDGTQAARDILTLNASEKLTHSGKSSAPSLSLSAPELTSSGVLVGS CdiA_EC 536 (578) QNAQLDGTQAARDILTLNASEKLTHSGKSSAPSLSLSAPELTSSGVLVGS CdiA_B_pseudo (611) NTVTNSGSMSADANLHVIGQSVDNGNGALHAGQTTTVDAGNHLSNAGGRV CdiA_Y_pestis (613) GDIALTGATVMAANTLFADTRQILRSDKAYLTADQINLTAYSLSNVEGRV CdiA_pertussis (643) RSRGAVTVSGGGAVNLGDVQSDGQVRATSAGAMTVRDVAAAADLALQAGD 701 750 CdiA_K93 (628) ALN----------------------------------------------- CdiA_EC 536 (628) ALN----------------------------------------------- CdiA_B_pseudo (661) EGQS---------------------------------------------- CdiA_Y_pestis (663) VQKG---------------------------------------------- CdiA_pertussis (693) ALQAGFLKSAGAMTVNGRDAVRLDGAHAGGQLRVSSDGQAALGSLAAKGE 751 800 CdiA_K93 (631) -------------------------------------------------- CdiA_EC 536 (631) -------------------------------------------------- CdiA_B_pseudo (665) -------------------------------------------------- CdiA_Y_pestis (667) -------------------------------------------------- CdiA_pertussis (743) LTVSAARAATVAELKSLDNISVTGGERVSVQSVNSASRVAISAHGALDVG 801 850

17

CdiA_K93 (631) ---------------TQSQTLTNSGLLQGKASLTVNTQRLDNQQNGTLYS

CdiA_EC 536 (631) ---------------TQSQTLTNSGLLQGEASLTVNTQRLDNQQNGTLYS CdiA_B_pseudo (665) ---------------AVLNGATLDNSQGTVNAATVSLNGTTLLNHGGTVT CdiA_Y_pestis (667) ---------------SGDFRLDLPGYLDNRGGVLLTKGNLALQAERLTSN CdiA_pertussis (793) KVSAKSGIGLEGWGAVGADSLGSDGAISVSGRDAVRVDHARSLADISLGA 851 900 CdiA_K93 (666) AADLTLDIPDIRNSGLITGDNGLMLNAVSLSNPGKIIADTLSVRATTLDG CdiA_EC 536 (666) AADLTLDIPDIRNSGLITGDNGLMLNAVSLSNPGKIIADTLSVRATTLDG CdiA_B_pseudo (700) QTGTGPMTVAITDTLDNSNNGLIQTRSTDLSLTSTTLINDNGGTITHVGP CdiA_Y_pestis (702) SQSLLGAGIQADGSKASKGDLQANTTQALIAQGQNVAAGTMTLSGSRVDL CdiA_pertussis (843) EGGATLGAVEAAGSIDVRGGSTVAANSLHANRDVRVSGKDAVRVTAATSG 901 950 CdiA_K93 (716) DGLLQGAGALALAGDTLSLGSNGRWLTAGDLSLRGKTLHTAGTTQGQNLT CdiA_EC 536 (716) DGLLQGAGALALAGDTLSQGSHGRWLTADDLSLRGKTLNTAGTTQGQNIT CdiA_B_pseudo (750) GTLMVGNGSGTVSNKAGAIASNGRTVLQGKTIDNSAGSASGQTGLSVNAA CdiA_Y_pestis (752) TGSQTHASNITITARDGDVTTREATLITPGTLSMTAVANPEQTLNNRGGK CdiA_pertussis (893) GGLHVSSGRQLDLGAVQARGALALDGGAGVALQSAKASGTLHVQGGEHLD 951 1000 CdiA_K93 (766) VQADRWANSGSVQATGN----------------LTASATGQLTSTGDIMS CdiA_EC 536 (766) VQADRWANSGSVLATGN----------------LTASATGQLTSTGDIMS CdiA_B_pseudo (800) DSITNLGGKLTSNANVDVTAGG-----------ALVNDGGELGSKTAATT CdiA_Y_pestis (802) LHADNIQLNLAKLENSNGEIAAATDLWLRLQSDFIHQAGARLTAGRDLLF CdiA_pertussis (943) LGTLAAVGAVDVNGTGDVRVAKLVSDAGADLQAGRSMTLGIVDTTGDLQA 1001 1050 CdiA_K93 (800) QGDTTLNAATTDNRGSLLSAGTLSLDGNSLDNSGTVQGNHVTIRQNGVTN CdiA_EC 536 (800) QGDTTLKAATTDNRGSLLSAGTLSLDGNSLDNSGTVQGDHVTIRQNSVTN CdiA_B_pseudo (839) IHSASLSNLNGKIVSPTLTATVAGLLDNSQNGDFEANQLALTAANLKNQG CdiA_Y_pestis (852) NSRGALINQYKLEAGRDMQLTALSIRNTSADRNTNADNSSLLAGRGLSLS CdiA_pertussis (993) RAQQKLELGSVKSDGGLQAAAGGALSLAAAEVAGALELSGQGVTVDRASA 1051 1100 CdiA_K93 (850) SGTLTGIAALTLAARMDMAS------------------PQPALMNNGGSL CdiA_EC 536 (850) SGTLTGIAALTLAARMVS--------------------PQPALMNNGGSL CdiA_B_pseudo (889) GHISQWQSGPTTLAVSGTLDN-----------------------SNGGVI CdiA_Y_pestis (902) TDSLFNRGAIYTTGVGQFTVNGNTENIGEIYTEQQLTFTATGNLANRGVM CdiA_pertussis (1043) SRARIDSTGSVGIGALKAGAVEAAS-------------PRRARRALRQDF 1101 1150 CdiA_K93 (882) LTSGDLTITAGSLANSG------AIQAADSLTARLTGELVSTAGSKVTSN CdiA_EC 536 (880) LTSGDLTITAGSLVNSG------AIQAADSLTARLTGELVSTAGSKVTSN CdiA_B_pseudo (916) QTNSTDLTLAPAVLDNSKGTITHGGTGTLTLTPGNGAGALQNTGGTIGTN CdiA_Y_pestis (952) QTRGEMQLSAQGDVNNSG----MLYSAGDQMRLSIAGNLTNEGKLHVANG CdiA_pertussis (1080) FTPGSVVVRAQGNVTVGRGDPHQGVLAQGDIIMDAKGGTLLLRNDALTEN 1151 1200 CdiA_K93 (926) GEMALSALNLSNSGQWIAKNLTLKANSLTSAGDITGVDALTLTVNQTLN- CdiA_EC 536 (924) GEMALSALNLSNSGQWIAKNLTLKANSLTSAGDITGVDTLTLTVNQTLN- CdiA_B_pseudo (966) GQAIVKAGSLDNGSGVIAAKLGLSATIAGAMNNTQGLMRSNAALSIIGNG CdiA_Y_pestis (998) EMRLLTEGNLDNRGSLYGAGNSDITTQGNAVNTGSVYTQGALQWLTKGS- CdiA_pertussis (1130) GTVTISADSAVLEHSTIESKISQSVLAAKGDKGKPAVSVKVAKKLFLNGT 1201 1250

18

CdiA_K93 (975) --------NHASGKLLSAGVLTLKADSVKNDGQLQGNATTITAGQLTNGG

CdiA_EC 536 (973) --------NQANGKLLSAGVLTLKADSVTNDGQLQGNATTITAGQLTNGG CdiA_B_pseudo (1016) ALSN----HQGHIEAGTPGDTSTLSIQAASIDNTDGAVHDFGTGKMTVQG CdiA_Y_pestis (1047) --------VRNSASIAALGDLQLRANDLLSDNQSLMAAGLKADGSRSDSG CdiA_pertussis (1180) LRAVNDNNETMSGRQIDVVDGRPQITDAVTGEARKDESVVSDAALVADGG 1251 1300 CdiA_K93 (1017) HLQGETLTLAASGGVNNRSGGVLMSRNALNVSTATLSNQGTIQGGGGVSL CdiA_EC 536 (1015) HLQGETLTLAASGGVNNRFGGVLMSRNALNVSTATLSNQGTIQGGGGVSL CdiA_B_pseudo (1062) GSQIVNSHAGGVDGMGQMTGQGDVTIGAASISNTQGGQLMGANLLIQGAT CdiA_Y_pestis (1089) NLAVSTEQALIAQGQNIAAGSLALAGSQIDLTGSQTQANAISLTAKSGDI CdiA_pertussis (1230) PIVVEAGELVSHAGGIGNGRNKENGASVTVRTTGNLVNKGYISAGKQGVL 1301 1350 CdiA_K93 (1067) NATDRLQNDGKILSGSNLTLTAQVLANTGSGLVQAATLLLDVVNTVNGGR CdiA_EC 536 (1065) NVTDRLQNDSKILSGSNLTLTAQVLANTGSGLVQAATLLLDVVNTVNGGR CdiA_B_pseudo (1112) LDNSGGQVGNVANATGDVNVAMSGAVTNTNGSITSTRDLSVAASTLLGGG CdiA_Y_pestis (1139) TLTSAVIKAATQLLVTQLAATRSSFIPP-SSIPPSSTQSSSTQASASPSA CdiA_pertussis (1280) EVGGALTNEFLVGSDGTQRIEAQRIENRGTFQSQAPAGTAGALVVKAAEA 1351 1400 CdiA_K93 (1117) VLATGSADVKGTTLNNTGTFQGADLLVNYHTFSNSGTLLGTSGLGVKGSS CdiA_EC 536 (1115) VLATGSADVKGTTLNNTGTLQGADLLVNYHTFSNSGTLLGTSGLGVKGSS CdiA_B_pseudo (1162) AYSAARDAAINLQGDFTTTPQTQFNIGRDLTFTLPGTFANSANLQSVHNL CdiA_Y_pestis (1188) WLRTDKASLIADQLTFDVQALSNLGGVIAQTGATDFNLNLPGYLDNRGGT CdiA_pertussis (1330) IVHDGVMATKGEMQIAGKGGGSPTVTAGAKATTSANKLSVDVASWDNAGS 1401 1450 CdiA_K93 (1167) LLQNG--------------------------------------------- CdiA_EC 536 (1165) LLQHG--------------------------------------------- CdiA_B_pseudo (1212) TVNAGN-------------------------------------------- CdiA_Y_pestis (1238) ILSKGN-------------------------------------------- CdiA_pertussis (1380) LDIKKGGAQVTVAGRYAEHGEVSIQGDYTVSADAIALAAQVTQRGGAANL 1451 1500 CdiA_K93 (1172) ------------------------------------------------TG CdiA_EC 536 (1170) ------------------------------------------------TG CdiA_B_pseudo (1218) ----------------------------------------IVNTGAMTAG CdiA_Y_pestis (1244) ----------------------------------------VA-----IQA CdiA_pertussis (1430) TSRHDTRFSNKIRLMGPLQVNAGGPVSNTGNLKVREGVTVTAASFDNETG 1501 1550 CdiA_K93 (1174) RLYSAGNLLLDAQDFSGQGQVVATGDVTLKLIAALTNHGTLAAGKTLSVT CdiA_EC 536 (1172) RLYSAGNLLLDAQDFSGQGQVVATGDVTLKLIAALTNHGTLAAGKTLSVT CdiA_B_pseudo (1228) SLLSTHSGDLTNYGAMVGGSVAIQASGTVSNLGPVALIGASDTSGLLEIV CdiA_Y_pestis (1249) QGLDSDSGSLLGAGVQSDGKLTNAGDLAVTVRQDLIAHGQSLAAGAMTLT CdiA_pertussis (1480) AEVMAKSATLTTSGAARNAGKMQVKEAATIVAASVSNPGTFTAGKDITVT 1551 1600 CdiA_K93 (1224) SQNAVTNGGVMQGDAMVLGAGEAFTNNGTLTAGKGNSVFSAQRLFLNAPG CdiA_EC 536 (1222) SQNAITNGGVMQGDAMVLGAGEAFTNNGMLTAGKGNSVFSAQRLFLNAPG CdiA_B_pseudo (1278) AHDIENRDDTTLGDSMPTTTIFGLGKVALAGGKDANGNYTNAALINNSSA CdiA_Y_pestis (1299) GSGVDLTGSQTQARGITITANKGDVSTQRANILSLGSLAINAGANAGQTL CdiA_pertussis (1530) SRGGFDNEGKMESNKDIVIKTEQFSNGRVLDAKHDLTVTASGQADNRG-- 1601 1650

19

CdiA_K93 (1274) SLQAGGDVSLNSRSDITISGFTGTAGSLTMNVAGTLLNSALIYAGNNLKL

CdiA_EC 536 (1272) SLQAGGDVSLNSRSDITISGFTGTAGSLTMNVAGTLLNSALIYAGNNLKL CdiA_B_pseudo (1328) AIQSGASMELHADKVTNTRRVMQTSGNTSQVDPALLQQLGISMSGCAAYY CdiA_Y_pestis (1349) NNQGGSLQANNIALNLGQLDNRTGKIAASQDLVLGLQSDFNILADSTLQA CdiA_pertussis (1578) S--LKAGHDFTVQAQRIDNSGTMAAGHDATLKAPHLRNTGQVVAGHDIHI 1651 1700 CdiA_K93 (1324) FTDRLHNQHGDILAGNSLWVQKDSSGTANSEIIN----------RSGNIE CdiA_EC 536 (1322) FTDRLHNQHGDILAGNSLWVQKDASGGANTEIIN----------TSGNIE CdiA_B_pseudo (1378) IAACSGQDVHWINLFHDPNYPDYDPAPIIAALKLQ--------------- CdiA_Y_pestis (1399) GRDFSFTTHGALTNDGQLLAGRKLSTRSNSLLNNGNIRAVQADLRASGAL CdiA_pertussis (1626) INSAKLENTGRVDARNDIALDVADFTNTGSLYAEHDATLTLAQGTQRDLV 1701 1750 CdiA_K93 (1364) TTRGDITMNTAHLLNSWD---------------------AISASHEVIPG CdiA_EC 536 (1362) THQGDIVVRTGHLLNQREGFSATTTTRTNPSSIQGMGNALVDIPLSLLPD CdiA_B_pseudo (1413) -------------------------------------------------- CdiA_Y_pestis (1449) TNRGEILTRGGLSTDANTLFNSGTLIGATATLNARERITNSGPNALIGAT CdiA_pertussis (1676) VDQDHILPVAEGTLRVKAKSLTTEIETGNPGS--LIAEVQENIDNKQAIV 1751 1800 CdiA_K93 (1393) SSHGVISPVPENNRWWGVVRHDGVEYLAVYWGKGATVPDEYRIRTGDTE- CdiA_EC 536 (1412) GSYGYFTREVENQHGTPCNGHGACNITMDTLYYYAPFADSATQRFLSSQN CdiA_B_pseudo (1413) -----------------------PGGVFTVPPNGGQWNSGYQYTTYEGKA CdiA_Y_pestis (1499) DKNGTLALLAPVIENSDTVTRTDTAPTTTLLGMGKVILAGGQDNSGNYS- CdiA_pertussis (1724) VGKDLTLSSAHGNVANEANALLWAAGELTVKAQNITNKRAALIEAGGNAR 1801 1850 CdiA_K93 (1442) -TVTVSASGHAARISGGADMHIRAGRLDNEASFILAGGGMTLSGDTLNNQ CdiA_EC 536 (1462) ITTVTGADNPAGRIASGRNLSAEAERLENRASFILANGDIALSGRELSNQ CdiA_B_pseudo (1440) TANTVTKLSPGAQIASGGDLDASTVKTFQNYWSSVTAAGNIKQPASLDMD CdiA_Y_pestis (1548) --SAAQVLNLSGLIESGNDLLVYAKTLTNRRQILTATTDFIVGDTETGAA CdiA_pertussis (1774) LTAAVALLNKLGRIRAGEDMHLDAPRIENTAKLSGEVQRKGVQDVGGGEH 1851 1900 CdiA_K93 (1491) GWQEGTTGKETVWRL----------ASGSLPKAWFTEPWYKVYRQVSPDA CdiA_EC 536 (1512) SWQTGTENEYLVYRYDPKT-FYGSYATGSLDKLPLLSPEFENNTIRFSLD CdiA_B_pseudo (1490) GWGATGQQAPGVTVVYSG----YYHYNNYDNSEHNWTLPFGDKPFVGGPG CdiA_Y_pestis (1596) YWTAENPDIPGGRYTQPPA-G-GPMNSDYIGTNYTSTVAYNRIDQISPEA CdiA_pertussis (1824) GRWSGIGYVNYWLRAGNGKKAGTIAAPWYGGDLTAEQSLIEVGKDLYLNA 1901 1950 CdiA_K93 (1531) TEASGTSPAGQYRAVISAAGDVSASFATDTGNTTVMPRAGGA-------- CdiA_EC 536 (1561) GREKDYTPGKTYYSVIQAGGDVKTRFTSSINNGTTTAHAGSV-------- CdiA_B_pseudo (1536) GYTQAAPADVRQYSLPDYRSTWGANGTISGNGVSVNNTAANATIP----- CdiA_Y_pestis (1644) QLLAGGNLTLQVGTLENNWSKVSAQGVIDLTGVTLQQDDWGSQQRLVEQT CdiA_pertussis (1874) GARKDEHRHLLNEGVIQAGGHGHIGGDVDNRSVVRTVSAMEYFKTPLP-- 1951 2000 CdiA_K93 (1573) --------------------------------GNTITVPSLNSLTPPTVS CdiA_EC 536 (1603) --------------------------------SPVVSAPVLNTLSQQTGG CdiA_B_pseudo (1581) -------------------------------------------------- CdiA_Y_pestis (1694) TSSGEYRYRTYKGKLWGIAWGPEMKLRLNNQYASSITAKTLTGSGTVINN CdiA_pertussis (1922) --------------------------------VSLTALDNRAGLSPATWN 2001 2050

20

CdiA_K93 (1591) QGVSGEALLNESGTGITGPVW--NDALPDTLKDIPGALSLSGAS--VSSY

CdiA_EC 536 (1621) DSLTQTALQQYEPVVVGSPQW--HDELAGALKNIAGGSPLTGQTGISDDW CdiA_B_pseudo (1581) ---S--------------------------LGLLPGQAVPGLTIGTVSGN CdiA_Y_pestis (1744) TVINNGAAPGAIVAPRDRDST--GKNIAVEFNGIALTLPRSGLYQLKTDK CdiA_pertussis (1940) FQSTYELLDYLLDQNRYEYIWGLYPTYTEWSVNTLKNLDLGYQAKPAPTA 2051 2100 CdiA_K93 (1637) PLPSGNNGYFVPSTDPDSPYLITVNPKLDGLGKVDSSLFAGLYDLLRMQP CdiA_EC 536 (1669) PLPSGNNGYLVPSTDPDSPYLITVNPKLDGLGQVDSHLFAGLYELLGAKP CdiA_B_pseudo (1602) ASGTQSGAAAIKGGTPTWVDPVIASATAVNVLSNLTIPQGGLYRPNSAPN CdiA_Y_pestis (1792) GDYAPGPEAALSLANISPPSSLDATGPRGVPPPSDDLNRTGLVTPDRAVS CdiA_pertussis (1990) PPMPKAPELDLRGHTLESAEGRKIFGEYKKLQGEYEKAKMAVQAVEAYGE 2101 2150 CdiA_K93 (1687) GEAPRETDPAYTDEKQFLGSSYILDRLGLKPEKDYRFLGDAAFDTRYVSN CdiA_EC 536 (1719) GQAPRETAPSYTDEKQFLGSSYFLDRLGLKPEKDYRFLGDAVFDTRYVSN CdiA_B_pseudo (1652) PTYLIETNPAFTRMNNFLSSDYYLNQIGVNPLTTEKRLGDGFYEQQLVRN CdiA_Y_pestis (1842) GGYLVETHPAFASLNNWKGSDLYLQQLSSDPSVIHKRLGDNAYEQRLLRD CdiA_pertussis (2040) ATRRVHDQLGQRYGKALGGMDAETKEVDGIIQEFAADLRTVYAKQADQAT 2151 2200 CdiA_K93 (1737) VILNQTGSRYINGTGSDLAQMKYLMDSAAAQQKALGLTFGVSLTAGQVAQ CdiA_EC 536 (1769) AVLSRTGSRYLNGLGSDTEQMRYLMDNAARQQKGLGLEFGVALTAEQIAQ CdiA_B_pseudo (1702) QVTQLTGKAVLGPYTDLQGMYQSLMLAGAELSKSLNLPLGMSLSAQQVAA CdiA_Y_pestis (1892) QVLALTGRTVASDYRSEQAQFEQLFAAGVQYSKAFNLAPGTRLSAEQMAT CdiA_pertussis (2090) IDAETDKVAQRYKSQIDAVRLQAIQPGRVTLAKALSAALGADWRALGHSQ 2201 2250 CdiA_K93 (1787) LTRSLLWWESVTINGQTVMVPKLYLS---PEDITLHNGSVISGNNVQLAG CdiA_EC 536 (1819) LDGSILWWESATINGQTVMVPKLYLS---PEDITLHNGSVISGNNVQLAG CdiA_B_pseudo (1752) LTTNVIIMQTETVGGQQVLVPVVYLAK--ADQQNANGPLITAGNIDLKNT CdiA_Y_pestis (1942) LTGNIVLMENRDVAGQTVLVPVVYLAGVKPGDLRANGALIAAENISLTEV CdiA_pertussis (2140) LMQRWKDFKAGKRGAEIAFYPKEQTVLAAGAGLTLSNGAIHNGENAAQNR 2251 2300 CdiA_K93 (1834) GNITNSGSSINAQNDLLLDRTGSIDNLNAGLINAGGALNLKAIGDIGNIS CdiA_EC 536 (1866) GNITNSGSSINAQNGLSLDSTGYIDNLNAGLISAGGSLDLSAIGDISNIS CdiA_B_pseudo (1800) QVFTNSGTVKADTTLALQ---GKQIDNAFGALQSGGLTSLDTTGNVDLTS CdiA_Y_pestis (1992) QGFANAGAISASNNLQISMAKDITLNNRCGLLQAGNHLQLSTLNSDIDLT CdiA_pertussis (2190) GRPEGLKIGAHSATSVSGSFDALRDVGLEKRLDIDDALAAVLVNPHIFTR 2301 2350 CdiA_K93 (1884) SVISGKTVSLESATGNISNLTRTEQWAM----NNGYNHFSGTDTGPLAAV CdiA_EC 536 (1916) SVISGKTVQLESVSGNISNITRRQQWNAGSDSRYGGVHLSGTDTGPVATI CdiA_B_pseudo (1847) ANVKAGSLDLNAGNKLILDTATQTTHQVS----RDGATSDKTTLGPAANL CdiA_Y_pestis (2042) SARLNATNLQLDSGRDVILRTASDQYSS----GNGAVQRTQTILGPLASL CdiA_pertussis (2240) IGAAQTSLADGAAGPALARQARQAPETDG--MVDARGLGSADALASLASL 2351 2400 CdiA_K93 (1930) RATDSLFMGAAGDISITGAAVS---------------------------- CdiA_EC 536 (1966) KGTDSLSLDAGKNIDITGATVSSGGTLGMSAGNDINIAANLISGSKSQSG CdiA_B_pseudo (1893) NVAGDASIKTGGDFQQNAGNLS---------------------------- CdiA_Y_pestis (2088) NISNNAVITAQRDFIQQGAGIN---------------------------- CdiA_pertussis (2288) DAAQGLEVSGRRNAQVADAGLAGPSAVAAPAVGAADVGVEPVTGDQVDQP 2401 2450

21

CdiA_K93 (1952) --------------------------------------------------

CdiA_EC 536 (2016) FWHTDDNSASSTTSQGSSISAGGNLAMAAGHNLDVTASSV---------- CdiA_B_pseudo (1915) -------------------------------------------------- CdiA_Y_pestis (2110) -------------------------------------------------- CdiA_pertussis (2338) VVAVGLEQPVATVRVAPPAVALPRPLFETRIKFIDQSKFYGSRYFFEQIG 2451 2500 CdiA_K93 (1952) --AGDSVLLAAGNDLNMNAIQAGERRRYGGSGWYETHAVAPTVTAGNSLM CdiA_EC 536 (2056) -SAGHSALLSAGNDLSLNAVRESKNSRNGRSESHESHAAVSTVTAGDNLL CdiA_B_pseudo (1915) --VGGNLNANIGGNWNLGVQQTGEHKVVQRANGVSDTDLNSATGSTVNVG CdiA_Y_pestis (2110) --IGKDLQVNTGGDWLLSTVQRSDQISAQYGGGSATSGSLRHLGSEVKVG CdiA_pertussis (2388) YKPDRAARVAGDNYFDTTLVREQVRRALGGYESRLPVRGVALVAKLMDSA 2501 2550 CdiA_K93 (2000) L----SAGRDVNSQAAGITAENSMDIRAGRDVNMAAESTGAGDHDSTFSM CdiA_EC 536 (2105) L----VAGRDVASQAAGVAAENNVVIRGGRDVNLVAESAGAGDSYTSKKK CdiA_B_pseudo (1963) GKSAIGVGGDLTAQGARLDFGQGGTVAAKGNVTFGAASTTSTINANSSGD CdiA_Y_pestis (2158) G----ALSANVDNLTAVGARVNAGTIDVRAQNITLSAATDSLSVTGGSSS CdiA_pertussis (2438) GTVGKALGLKVGVAPTAQQLKQADRDFVWYVDTVIDGQKVLAPRLYLTEA 2551 2600 CdiA_K93 (2046) KTVHDSV--------RQQGTDMTSGGDITVTAGRDITSVATAVTAKG--- CdiA_EC 536 (2151) KEINETV--------RQQGTEIASGGDTTVNAGRDITAVASSVTATG--- CdiA_B_pseudo (2013) QGNRSYAETRHGADQALTGTTVKGGDTLNVVSGKDINVIGSTIDLKKG-- CdiA_Y_pestis (2204) KRHTSSVNLYD---ETLLGSQLNATGDINLQTVNDITLSASAVQTDG--- CdiA_pertussis (2488) TRQGITDQYAGGGALIASGGDVTVNTDGHDVSSVNGLIQGRSVKVDAGKG 2601 2650 CdiA_K93 (2085) --------DIRVNAGHDIVLGTATESDYHYSESGETRNRLLSHQTTRTIT CdiA_EC 536 (2190) --------NISVNAGRDVALTTATESDYHYLETKKKSGGFLSKKTTHTIS CdiA_B_pseudo (2061) --------DANLLAAGDVNVGAATETHVYNSRETHSRSGVVSGTKIASSQ CdiA_Y_pestis (2248) --------ALKLAAGGDVTLTSQTEQHDEQRNHTGTKKGLLSSTTTRSEE CdiA_pertussis (2538) KVVVADSKGAGGGIEADDEVDVSGRDIGIEGGKLRGKDVRLKADTVKVAT 2651 2700 CdiA_K93 (2127) EDSVTREKGSLLSGNRVTVNAGNNLTVQGSDVVADRDVSLAADNHVDVLA CdiA_EC 536 (2232) EDSASREAGSLLSGNRVTVNAGDNLTVEGSDVVADQDVSLAAGNHVDVLA CdiA_B_pseudo (2103) DATSTVANGSLISADGVSIGSGKDINVQGSTVVGTHDVALNAAHDVNITT CdiA_Y_pestis (2290) GRSQTLAVGSMLSAGSIDVSS-QNIAVAGSSVVADKDIRLRAQENLTVST CdiA_pertussis (2588) SMRYDDKGRLAARGDGALDAQGGQLHIEAKRLETAGATLKGGKVKLDVDD 2701 2750 CdiA_K93 (2177) ATSTDTSWRFKETKTSGLTGTGGIGFTTGSSKTTHDRREAGTTQSQSAST CdiA_EC 536 (2282) ATSTDTSWRFKETKKSGLMGTGGIGFTIGSSKTTHDRREAGTTQSQSAST CdiA_B_pseudo (2153) SQDTSQSSTTYQEQHSGLMSGGGLSFSVGNSK--LAQQNQSSSVTNNAST CdiA_Y_pestis (2339) AQQSESGSQLFEQKKSGLMSTGGIGVFIGTSR--QKTTDQTQTVSHVGST CdiA_pertussis (2638) VKLGGVYEAGSSYENKSSTPLGSLFAILSSTTETNQSAHANHYGTRIEAG 2751 2800 CdiA_K93 (2227) IGSTAGNVSITAGKQAHISGSDVIANRDISITGDSVVVDPGHDRRTVDEK CdiA_EC 536 (2332) IGSTAGNVSITAGKQAHISGSDVIANRDISITGDSVVVDPGHDRRTVDEK CdiA_B_pseudo (2201) VGSVDGNLTVNAGNTLHVKGSDLVAGKDVTGTAANIVVDSATDTTRQAQQ CdiA_Y_pestis (2387) VGSLTGNVRLEAGNQLTLHGSDVVAGKDLALTGADVAISAAENSRSQQYT CdiA_pertussis (2688) TLEGKMQNLEIEGGSVDAAHTDLSVARDARFKAAADFAHAEHEKDVRQLS 2801 2850

22

CdiA_K93 (2277) FEQKKSGLTVALSGAVGSAINNAVTMAREAKETSDSRLAALKGTQAVLSG

CdiA_EC 536 (2382) FEQKKSGLTVALSGTVGSAINNAVTSAQETKESSDSRLKALQATKTALSG CdiA_B_pseudo (2251) QQTSKSGLTVGLSGSVGDAINNAISETQAARESAKDSNGRASALHSIAAA CdiA_Y_pestis (2437) AESKQRGLTVALSGPVGSAVNTAVTTAKAAREENTGRLAGLQGVKAALSG CdiA_pertussis (2738) LGAKVGAGGYEAGFSLGSESGLEAHAGRGMTAGAEVKVGYRASHEQSSET 2851 2900 CdiA_K93 (2327) VQAGVNHGLQQQSAD-PNN----GIGVSISLNHQQSKSETKYQHDIVSGS CdiA_EC 536 (2432) VQAGQAATMASATGD-PN-----ATGVSLSLTTQKSKSQQHSESDTVSGS CdiA_B_pseudo (2301) GDVAFGGLGAKALLDGAKGPQAPSIGVQVSVGSSHSSMQSSEDQTIQRGS CdiA_Y_pestis (2487) VQAVQAGQLVQAQGGGITE----MVGVSVSLGSQKSSSQQQQEQTQVSGS CdiA_pertussis (2788) EKSYRNANLNFGGGSVEAGNVLDIGGADINRNRYGGAAKGNAGTEEALRM 2901 2950 CdiA_K93 (2372) TLSAGNNVSVTATGKNKDHNNSGDMLITGSQIKSGNDTSLNAQNDILLAA CdiA_EC 536 (2476) TLNAGNNLSVVATGKNRG-DNRGDIVIAGSQLKVGGNTSLDAANDILLSG CdiA_B_pseudo (2351) SINAGGNAKLIATGNG---TPKDGNITIAGSNVNAANVALVANNQVNLVN CdiA_Y_pestis (2533) ALTAGNNLSIKASG--------SDILIAGSQLKAGGDTRLDAARDVQLLG CdiA_pertussis (2838) RAKKVESTKYVSEQTSQS-SGWSVEVASTASARSSLLTAATRLGDSVAQN 2951 3000 CdiA_K93 (2422) AADTRQTTGKNSSKGGGVGVSFGGGTNGGGLSIFAGINGSEGREKGNGTT CdiA_EC 536 (2525) AANTQKTTGRNSSSGGGVGVSIGAGGNGAGISVFAGVNAAKGSEKGNGTE CdiA_B_pseudo (2398) TTDTDKTQSSNSSSGSSVGVSIG----TNGIGVSASMQRAHGDGNSDAAI CdiA_Y_pestis (2575) AANRQKTDGSNSSRGGSVGVSVG----GSGLSVFANANKGQGNERGDGTF CdiA_pertussis (2887) VEDGREIRGELMAAQVAAEATQLVTADTAAVALSAGISADFDSSHSRSTS 3001 3050 CdiA_K93 (2472) WTETTLDAGKNVSLTSGRDTTLSGAQVSGEKVTADVGNNLTISSLQDSDR CdiA_EC 536 (2575) WTETTTDSGKTVTINSGRDTVLNGAQVNGNRIIADVGHDLLISSQQDTSK CdiA_B_pseudo (2444) QNNTHINASQTATIVSGGDTNVIGANVNANKVVADVGGNLNVASVQDTTV CdiA_Y_pestis (2621) WTETTVDSGGMFSLRSGRDTALTGAQVSAETVKADVGRNLTLQSQQDRDN CdiA_pertussis (2937) QNTQYLGGNLSIEATEGDATLVGAKFGGGDQVSLKAAKSVNLMAAESTFE 3051 3100 CdiA_K93 (2522) YDSRQNRVAAGGSFTFGSMSGSGYASISQDKIKSNYDSVREQSGIYAGKD CdiA_EC 536 (2625) YDSKQTSVAAGGSFTFGSMTGSGYIAASRDKMKSRFDSVAEQTGMFAGDG CdiA_B_pseudo (2494) SAAHQSSAGGG--FTISQTGGGASFSAQNGHADGNYAGVNEQAGIQAGSG CdiA_Y_pestis (2671) YDAKQSRASGGISVPVAGGGAAVNLSMSRDRLSSQYDSVQAQTGIFAGSG CdiA_pertussis (2987) SYSESHNFHASADANLGANAVQGAVGLGLTAGMGTSHQITNETGKTYAGT 3101 3150 CdiA_K93 (2572) GFDVTVGNHTQLNGAVIASTATDDKNSLNTNTLGWSDIHNQADYKASHTG CdiA_EC 536 (2675) GFDITVGRHTQLDGAVIASTATPDKNHLDTGTLGFSDLHNEADYKVSHSG CdiA_B_pseudo (2542) GFDVTVRGNTDLKGAYIASTADASKNSLTTGTLTTSDIENHSHYSANSAG CdiA_Y_pestis (2721) GVDIRVGEHTQLDGAVIASTAAADKNTLDTGTLGFSDIKNKAVFTVEHQG CdiA_pertussis (3037) SVDAANVSIDAGKDLNLSGSRVRGKHVVLDVEGDINATSKQDERNYNSSG 3151 3200 CdiA_K93 (2622) ISLSGGSGMSASQMVASNAIAGAANALTGMSGSSGHAEGTTSSAISGGNL CdiA_EC 536 (2725) ISLSGGGS------FGDKFQGNMPGGMISAGGHSGHAEGTTQAAVAEGTI CdiA_B_pseudo (2592) FSAGASVGVSTKAVGPSSVSGSGGVTPMVFQNDSGDQSATTKSAVSAGTI CdiA_Y_pestis (2771) GSLSTGGP------VGSDLLSNLGGMVLAGLGNGGYAEGTTQAAVSEGTI CdiA_pertussis (3087) GGWDASAGVAIQNRTLVAPVGSAGFNFNTEHDNSRLTNDGAAGVVASDGL 3201 3250

23

CdiA_K93 (2672) IIRNKESQKQDIAGLSRDPENANGSIAPIFDREKEQKRLQEAQVISQISG

CdiA_EC 536 (2769) TIRDRDNQKQNLANLSRDPAHTNDSISPIFDKEKEQRRLQTVGLISDIGS CdiA_B_pseudo (2642) NITKPGEQTQDVANLNRDTTNLNGTVSKTPDVQKMLSQQADTMNAAQAAG CdiA_Y_pestis (2815) TVRDTENQQQNVDDLSRDTGNANGSIGPIFDKEKEQNRLKEVQLIGEIGG CdiA_pertussis (3137) TGHVKGDANLTGATIADLSGKGNLKVDGAVNAQNLKDYRDKDGGSGGLNV 3251 3300 CdiA_K93 (2722) QMSNIVMTYGETEAMKAARKEH--------------------------PG CdiA_EC 536 (2819) QVADIARTQGELNALKAAQDKYGPVP---------------ADATEEQRQ CdiA_B_pseudo (2692) QTVSQAIGLYADHKRDAALDAADKAYKAG--------------------- CdiA_Y_pestis (2865) QALDIASTQGKIIATHAANDKMKAVKPEDIAAAEKQWEKAHPGKAATAED CdiA_pertussis (3187) GISSTTLAPTVGVAFGRVAGED---------------------------- 3301 3350 CdiA_K93 (2746) MSDAQLRETPEYREVMKGYGTGSTPQMVVQAITGVLGGLNAGNPGQVLAG CdiA_EC 536 (2854) AYLAKLRDTPEYKKEQEKYGTGSDMQRGIQAATAALQGLVGGNMAGALAG CdiA_B_pseudo (2721) ---DLAGAQAALNEAKGWMEGGASRAELQMGGGALIGGLGGGSALTAIGG CdiA_Y_pestis (2915) INQQIYQTAYNQAFNESGFGTGGPVQRGMQAATAAVQGLAGGNMGAALTG CdiA_pertussis (3209) ----------YQAEQRATIDVGQTKDPARLQVGGGVKGTLNQDAAQATVV 3351 3400 CdiA_K93 (2796) GLNPAVAQLI--KQATGDNREANLMAHAVWGALAAQLGGNNAASGAAGAF CdiA_EC 536 (2904) ASAPELANIIGHHAGIDDNTAAKAIAHAILGGVTAALQGNSAAAGAIGAG CdiA_B_pseudo (2768) AAGAGMSSLLANQAEKISKSVGDTTGSSLVGNIAANVAATVGGALVGGSA CdiA_Y_pestis (2965) ASAPYLAGVIK--QSTGDNPAANTMAHAVLGAVTAYASGNNALAGAAGAA CdiA_pertussis (3249) QRNKHWAGGGSEFSVAGKSLKKKNQVRPVETPTPDVVDGPPSRPTTPPAS 3401 3450 CdiA_K93 (2844) SGELAARYIIDNYYGGRT---DNLSEQERQQISMLATIASGIAGGLVGNS CdiA_EC 536 (2954) TGEVIASAIAKSLYPGVDP--SKLTEDQKQTVSTLATLSAGMAGGIASGD CdiA_B_pseudo (2818) GAAMASNVQLYNAGNDSN---NQTSNDVFASLSKKVAQAIAMTADGKAGV CdiA_Y_pestis (3013) TAELMAPTIISALGWDKN----TLTEGQKQAVSALSTLAAGLAGGLTGDS CdiA_pertussis (3299) PQPIRATVEVSSPPPVSVATVEVVPRPKVETGSAASASAGGAQVVPVTPP 3451 3500 CdiA_K93 (2891) TSAAGTGAQAGRNSVENNAMSGLEGFGTGFQSYVQAQEALVNNTNLTDKN CdiA_EC 536 (3002) VAGAAAGAGAGKNVVENNALSLVARGCAVAAPCRTKVAEQLLEIGAKAGM CdiA_B_pseudo (2865) WNGMVNVAGVIVNLPNGGPFASPGDPGYVSLDGLKKPYKSGTSIGPDAEF CdiA_Y_pestis (3059) TADALAGGQAGKNAVENNLLGGNEFTHTQFVQKHGADVLSCADNPSNAAC CdiA_pertussis (3349) KVEVAKVEVVPRPKVETAQPLPPRPVVAEKVTTPAVQPQLAKVETVQPVK 3501 3550 CdiA_K93 (2941) GKVLNPATPEEIKYASDKLVTGSIPEGQDPARGLLISWGAGASVFGGELI CdiA_EC 536 (3052) AGLAGAAVKDMADRMTSDELEHLITLQMMGNDEITTKYLSSLHDKYGSGA CdiA_B_pseudo (2915) WTPVLATLGLGGKAAAGTGATTTSADAATVGNGALKTASGDLSAAGNAAR CdiA_Y_pestis (3109) QRGIAENKAYIAALATGSVALLPGSSQAMWALGAGTNAGMQYADNGKINP CdiA_pertussis (3399) PETTKPLPKPLPVAKVTKAPPPVVETAQPLPPVKPQKATPGPVAEVGKAT 3551 3600 CdiA_K93 (2991) APAVGTVAVIGGTLLGGTTDAVKQFLTLKPGEQYSTTDTLIAAGEGGLTQ CdiA_EC 536 (3102) ASNPNIGKDLTDAEKVELGGSGSGTGTPPPSENDPKQQNEKTVDKLNQKQ CdiA_B_pseudo (2965) TQPYGNGASASPSPGTATAGSSGANAQLPTANGGVAAAGTSSATNVGKVV CdiA_Y_pestis (3159) VNSVAAGWINVITMGQGWKGTIAWNAAGGALINAINGGDPLTGAITNGTG CdiA_pertussis (3449) VTTVQVQSAPPKPAPVAKQPAPAPKPKPKPKPKAERPKPGKTTPLSGRHV 3601 3650

24

CdiA_K93 (3041) GKGVIFSTFINTMGAYLGSKAKGEDPTGPMVGNAIGTALGNKAGDKFTKE

CdiA_EC 536 (3152) ESAIKKIDNTIKNALKDHDIIGTLKDMDGKPVPKENGGYWDHMQEMQNTL CdiA_B_pseudo (3015) IDGKIGGQLEARGWTQQEVQAVVNEGPVGTTMDNRSAGKTPDGLPRNDSA CdiA_Y_pestis (3209) AGFGYGVGNYVVKPAANTLGKWITGGWNPKFDPNLLKYAEVKGQLGISKE CdiA_pertussis (3499) VQQQVQVLQRQASDINNTKSLPGGKLPKPVTVKLTDENGKPQTYTINRRE 3651 3692 CdiA_K93 (3091) MLSRGFGSVTSEVTGTVTGSVIGTVTDYQIEKLGKGNKEGAK CdiA_EC 536 (3202) RGLRNHADTLKNVNNPEAQAAYGRATDAINKIESALKGYGI- CdiA_B_pseudo (3065) SVYGSKSGYVVVNDRTGEVVQVSGKNDPGWIPDSRIKWK--- CdiA_Y_pestis (3259) MLPSKIPSAVGNAGGSLSSEFGSSLIQQKKDAMEDSK----- CdiA_pertussis (3549) DLMKLNGKVLSTKTTLGLEQTFRLRVEDIGGKNYRVFYETNK

three or . The roteins

Fig. S8. Complementation with the cdiA homologue from UPEC strain 536. The cdiA homologue in UPEC 536 was cloned into plasmid pCC1 under lac promoter control to generate plasmid pDAL665. Plasmid pDAL665 was then transformed into cdiA–B+ E. coli (EPI300 containing a stop codon mutation within cdiA) (S13) to test complementation. DL4793 target cell viability was determined in mixed cultures containing E. coli cdiA–B+ (pDAL665)(red closed squares) and controls containing either E. coli cdiA–B+ (black open triangle) or E. coli cdiA–B-(pDAL665) (closed black triangle).

Fig. S7. Sequence alignments of CdiA homologues. Alignment of CdiA from EC93 (GenBank accession number DQ100454) with homologues from UPEC536, pathogenicity island II (AJ494981); Burkholderia pseudomallei K96243 (BX571966); Yersinia pestis CO92 (AL590842) and Bordetella pertussis Tohama I (BX470248). Regions identical among all proteins are shown in yellow, regions identical betweenmore proteins are shown in blue, and amino acids with similar functional groups are shown in greenboxed region at the amino terminus of CdiA shows the TPS domain conserved in all TPS-secreted p(S12). The alignment was performed using the Vector NTI ALIGN program.

ig. S8. F

0 2 4 6104

105

106

Time (h)

Targ

et c

ells

(

107

108

109

cfu/

ml)

25

Table S1. Strains, Plasmids and Oligonucleotides Strain Relevant Reference or source characteristic(

ue-Dawley rat acZ∆M15 ∆lac

cZ∆M15 ∆lacX061 derivative)

1 flb5301 deoC1 ptsF25 rbsR4, proA2, lacY1,

derivative

GFP expre

s)

U1

74

ga

ssi

EC93 E. coli isolated from the feces of a Sprag This study

DH5α (NalR) F- supE44 hsdR17(rK-mK

+) recA1 φ80dl 69 gyrA96 endA1 thi-1 relA1 deoR λ- (S1)

EPI100 (StrR) F- mcrA ∆(mrr-hsdRMS-mcrBC) φ80dla recA1 endA1 araD139 ∆(ara, leu)7697 galU galK λ- rpsL nupG (MC1 Epicentre

EPI300 (StrR) EPI100 (trfA) strR Epicentre CAG18439 (TetR) MG1655 lacZU118 lacI42::Tn10 (S2) MC4100 (StrR) F- araD139 ∆lacU169 rpsL150 relA (S3)

HB101 (StrR) supE44, hsdS20(rB- mB

-), recA13, ara-1 lK2, rpsL20, xyl-5, mtl-1, leuB6, thi-1 λ- (S1)

UPEC A8 Human E. coli uropathogen isolate (S4) UPEC A50 Human E. coli uropathogen isolate (S4) UPEC J96 Human E. coli uropathogen isolate (S5) UPEC 3576H Human E. coli uropathogen isolate (S6) UPEC 536 (StrR) Human E. coli uropathogen isolate (S7) DL159 MC4100 containing pRS551 (S8) DL430 MC4100, NalR This study DL3852 Spontaneous rifampicin-resistant EC93 This study DL4527 EPI100/EC100 containing pWEB::TNC This study DL4577 EPI100/EC100 containing pDAL660∆1-39 This study DL4608 DH5� containing pDAL660∆1-39 This study DL4793 CAG18439 containing pWEB::TNC This study DL4905 MC4100-gfpmut3 containing pDAL660∆1-39 This study DL4920 CAG18439 containing pDAL672 This study

DL4955 MC4100papBAmut13-gfpmut3 (constitutive on) containing pDAL660∆1-39::FLAG1 This study

DL4956 EPI100 pDAL660∆2-63 This study DL4957 EPI300 pDAL660∆1-39::1-32TP- This study DL4958 EPI300 pDAL660∆1-39::2-7TP- This study

26

Plasmid Relevant characteristic(s) Reference or source

pANN801-13 EcoRI/BamHI fragment from UPEC strain 536 containing the sfa operon cloned into (S9) pBR322 , TetR S10)

pCC1 Cloning vector containing the E. coli F factor origin of r

replication, the oriV origin of replication and lac p omoter, chloramphenicol-resistant Epicentre

pDAL230B pBR325 containing pap-21 operon, expresses Pap-21 pili This study

pDAL655 pACYC184 containing a 13 kb BamH1 DNA fragment from UPEC C1212 encompassinthe pap-17 pili operon. Constitutively expresses Pap-17 pili

g This study

pDAL656 cdiB from E. coli A8 inserted into the Eco72I site of pCC1 under lac promoter control This study pDAL657 cdiB from E. coli A50 inserted into the Eco72I site of pCC1 under lac promoter control This study pDAL658 cdiB from E. coli J96 inserted into the Eco72I site of pCC1 under lac promoter control This study

Vector pWEB::TNC containing ~46 kb DNA fragment from EC93 including cdiABIpDAL660∆1-39 pDAL660 deletion containing 16,734 bp DNA including cdiABI This study pDAL660∆2-63 diABI insert deleted pDAL660 deletion with the entire c This study pGPS5 GPS-LS kanamycin-resistant Transprimer-5 donor, R6K origin of replication New England Biolabs pDAL660∆1-39::1-6TP- AL660∆1-39 at bp 7,559 Transprimer-5 15bp insertion in pD This study pDAL660∆1-39::1-8TP- AL660∆1-39 at bp 8,476 Transprimer-5 15bp insertion in pD This study pDAL660∆1-39::1-14TP- AL660∆1-39 at bp 9,467 Transprimer-5 15bp insertion in pD This study pDAL660∆1-39::1-23TP- in pDAL660∆1-39 at bp 9,445 Transprimer-5 15bp insertion This study pDAL660∆1-39::1-30TP- 5bp insertion in pDAL660∆1-39 at bp 4619 Transprimer-5 1 This study pDAL660∆1-39::1-32TP- t bp 2,423 Transprimer-5 15bp insertion in pDAL660∆1-39 a This study pDAL660∆1-39::1-33TP- 0∆1-39 at bp 808 Transprimer-5 15bp insertion in pDAL66 This study pDAL660∆1-39::1-39TP- 1-39 at bp 3,708 Transprimer-5 15bp insertion in pDAL660∆ This study pDAL660∆1-39::1-64TP- AL660∆1-39 at bp 10,740 Transprimer-5 15bp insertion in pD This study pDAL660∆1-39::1-67TP- L660∆1-39 at bp 5,888 Transprimer-5 15bp insertion in pDA This study pDAL660∆1-39::1-82TP- 39 at bp 3,799 Transprimer-5 15bp insertion in pDAL660∆1- This study pDAL660∆1-39::2-7TP- DAL660∆1-39 at bp 1,507 Transprimer-5 15bp insertion in p This study pDAL660∆1-39::2-17TP- Transprimer-5 15bp insertion in pDAL660∆1-39 at bp 1,798 This study pDAL660∆1-39::2-30TP- -5 15bp insertion in pDAL660∆1-39 at bp 1,105 Transprimer This study pDAL660∆1-39::FLAG1 me at the PmeI site of pDAL660∆1-39::1-64TP- (see table S2) FLAG-tag inserted in-fra This study pDAL660∆1-39::FLAG2 PmeI site of pDAL660∆1-39::1-14TP- (see table S2) FLAG-tag inserted in-frame at the This study pDAL660∆1-39::FLAG3 e PmeI site of pDAL660∆1-39::1-67TP- (see table S2) FLAG-tag inserted in-frame at th This study

pBR325 Cloning vector. AmpR, CamR (

pDAL660 This study

27

28

pDAL6 FLAG4 FLAG-tag inserted in-frame at ∆1-39::1-39TP- (see table S2) 60∆1-39:: the PmeI site of pDAL660 This study ∆1-39:: FLAG-tag inserted in-frame at the PmeI site of pDAL660∆1-39::2-17TP- (see table S This study

pDAL660∆1-39::FLAG6 2) FLAG-tag inserted in-frame at the PmeI site of pDAL660∆1-39::1-33TP- (see table S This study

pDAL662 cdiB from EC93, amplified with oligos 854 and 855, and blunt-cloned into the Eco72I site of pCC1 under lac promoter control This study

pDAL663 cdiB from human uropathogen Strain 536, amplified with oligos 854 and 855, and blunt-ontrol cloned into the Eco72I site of pCC1 under lac promoter c This study

pDAL664 cdiI from EC93, amplified with oligos 883 and 884, and blunt-cloned into the Eco72I site of pCC1 under lac promoter control This study

pDAL664-REV ite cdiI from EC93, amplified with oligos 883 and 884, and blunt-cloned into the Eco72I sof pCC1 in transcriptional orientation opposite to the lac promoter This study

pDAL665 cdiA from 536, amplified with oligos 875 and 877, and blunt-cloned into the Eco 72I site of pCC1 under lac promoter control This study

pDAL672 SphI/EcoRI fragment from pDsRed-Express cloned into pTTQ19 to generate a lacIq-repressed, IPTG-inducible DsRed expression vector This study

pRS551 Ampicillin, Kanamycin-resistant (S8) pSH2 fim operon from UPEC J96 cloned into pACYC184 (S11)

pWEB::TNC Cosmid vector containing Tn5 transposase recognition sequences, ampicillin and chloramphenicol-resistant Epicentre

Oligonucleotide Sequence Refe urce rence or so854 5' CCCACGAGGAGAATATGC 3' This study 855 5' AACTCAGCAGGCGGTAAG 3' This study 865 5' GACTACAAGGACGACGATGACAAG 3' This study 867 5' AGACTACAAGGACGACGATGACAAGGC 3' This study 875 5' CAACTGCTGTAAGCGGAT 3' This study 877 5' ATCTCACGGTTTACTGGC 3' This study 883 5' AACCGCACAGCCTCTTGCTA 3' This study 884 5' GCTTTGGCTCTGTCACATCAGA 3' This study pWEB::TNC Sequencing Primer 5' TGTGAAATTTGTGATGCTATTGCT 3' Epicentre

N 5' ACTTTATTGTCATAGTTTAGATCTATTTTG 3' New England Biolabs S 5' ATAATCCTTAAAAACTCCATTTCCACCCCT 3' New England Biolabs

pDAL660 FLAG5 2)

Table S2. Mapping and analysis of insertion sequences within cdiAB

n M ion m g.

yp cs f

o bp Insertions1 Acids

Inserted

AG-tag(FLAG = DYKDDDDK) Reading

Frame CDI IMM2

11nu3A 2 Cde

5 bmb.

DIpen

ase-paering

= condent g

ir inseis rela

tact-drowth

rtionstive to

epend inhib

were the fi

ent inition (

constrrst bas

hibitiosee M

ucted e of th

n of gateria

as de c

rowls a

escodi

th nd M

ribed ing regi

phenotethod

at of

e, anor a

eriacdiB

d Issa

ls a. A

MMys)

nd ll

= .

Meinse

imm

thortio

un

ds. ns

ity

Bas are

to c

e-paapp

onta

r ed i

t-

n Fi

Location f 15 Amino FL insertions Open 2

1105 V P NA CdiB - + G (STO ) 1507 FK N CdiB - + V QN A

1798 F # DYKDDDDKKHF CdiA + + M KHF 5: MF

2423 ( P N CdiA - + TV STO ) A

3708 FK # DYKDDDDKKQQ CdiA + + V QQ 4: VF

3799 ( N CdiA - + PV STOP) A

4619 V N CdiA - + N (STOP) A

5888 N # DY DKANIT CdiA + CL IT 3: CL KDDD +

7559 V iA - V (STOP) NA Cd +

8476 V iA - N (STOP) NA Cd +

9445 NV iA (STOP) NA Cd - +

9467 CLNKQ #2: CLDY DKAKDDD NKQ CdiA + +

10740 CLNKL #1: CLDYKDDDDKANKL CdiA - +

808 LFKQQ #6: LFDYKDDDDKKQQ CdiB + +

29

30

Supp

1. D. Hanahan, J Mol Biol 166, 557-580 (1983). S2. M. Singer et al., Microbiol Rev 53, 1-24 (1989). S3. M. J. Casadaban, J Mol Biol 104, 541-555 (1976). S4. P. O'Hanley et al., N Engl J Med 313, 414-420 (1985). S5. en, S. Falk od Nut 75-89 (1983). S6. A. B G. V. L Rub m 601 988). S7. J. Hacker, S. Knap bel, J Bacteriol 154, 1145-1152 (198S8. R. W. Simons, F. Houman, N. Kleckner, Gene 53, 85-96 (1987). S9. J. Hacker et al., un 4S10. P. Prentki, F. Karch, S. Iida, J. Meyer, Gene 89-299 (1981). S11. R. A. Hull, R. E. Gill, P. Hsu, B. H. Minshew, S. Falkow, Infect Immun 33, 933-938 (1981). S12. B. Clantin et al. l Acad Sci U S A 1 194-6199 (2004)S13. S. K. Aoki and D.A. Low, unpublished data

lementary References

S

C. Svanborg EdD. A. Low, B.

R. Hull,raaten,

p, W. Goe

ow, H. Leffler, Prog Foing, D. L. Johnson, A. L.

r Sci 7, y, Infect Im

3). un 56, 2 -2609 (1

Infect Imm 7, 434-440 (1985). 14, 2

, Proc Nat 01, 6 .