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Supplemental Material

The second messenger c-di-GMP regulates Clostridium difficile toxin

production by controlling expression of sigD

Running Title: c-di-GMP represses Clostridium difficile toxins

Robert W. McKee*, Mihnea R. Mangalea*, Erin B. Purcell, Erin K. Borchardt and Rita

Tamayo

*Authors contributed equally to this work

Department of Microbiology and Immunology,

University of North Carolina at Chapel Hill, Chapel Hill, NC

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Table S1. Strains and Plasmids Used in this Study Name Description Reference E. coli DH5α F- φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1

hsdR17(rκ -, mκ+) phoA supE44 thi-1 gyrA96 relA1 λ- tonA

Invitrogen, (1)

HB101 F− mcrB mrr hsdS20(rB− mB

−)recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20

(2)

RT730 HB101(pRK24) pSigD This study RT465 HB101(pRK24) pMC-Pcpr (3) RT812 HB101(pRK24) pBL100::tcdR::ermB This work C. difficile 630 Wild type (4) JIR8094 (630E) Erythromycin-sensitive derivative of 630 (5) 630Δerm Erythromycin-sensitive derivative of 630 (6) RT476 630 pMC-Pcpr (3) RT477 630 pDccA (3) RT540 630 pDccAmut (3) RT731 630 pSigD This study RT744 JIR8094 pMC-Pcpr This study RT747 JIR8094 pSigD This study RT854 630Δerm tcdR::ermB This study RT856 630Δerm tcdR::ermB pMC-Pcpr This study RT858 630Δerm tcdR::ermB pSigD This study B. subtilis 1A716 BG-2 trpC2 sigD::pLM5 (7) MC190 trp- sacA::cprR,cprK lacA::tet amyE::Pcpr::lacZ (8) MC202 trp- sacA::cprR,cprK lacA::tet amyE::spec (8) RT838 trp- sacA::cprR,cprK lacA::tet amyE::spec sigD::pLM5 trpC2 This work RT839 trp- sacA::cprR,cprK lacA::tet sigD::pLM5 trpC2

amyE::PtcdA::lacZ Pcpr::sigD This work

RT840 trp- sacA::cprR,cprK lacA::tet sigD::pLM5 trpC2 amyE::PtcdB::lacZ::Pcpr::sigD

This work

RT841 trp- sacA::cprR,cprK lacA::tet sigD::pLM5 trpC2 amyE::PtcdR::lacZ::Pcpr::sigD

This work

RT842 trp- sacA::cprR,cprK lacA::tet sigD::pLM5 trpC2 amyE::PflgM::lacZ::Pcpr::sigD

This work

Plasmids pRK24 Broad host-range plasmid of Inc-P1 (9, 10) pMC123 E. coli- C. difficile shuttle vector; AmpR, CmR (1) pMC-Pcpr cprABC promoter cloned into pMC123, for nisin-inducible

expression (3)

pBL64 pCR2.1- intron template part A (12) pBL65 pCR2.1- intron template part B (12) pBL100 Targetron vector containing un-targeted group II intron (12) pDccA pMC-Pcpr::dccA (3) pDccAmut pMC-Pcpr::dccA(AADEF) (3) pSigD pMC-Pcpr::sigD This work pHK23 E. coli plasmid for integration of lacZ transcriptional fusions at

the amyE locus in B. subtilis (13)

pRT822 pHK23::PtcdA::lacZ This work pRT823 pHK23::PtcdB::lacZ This work

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pRT824 pHK23::PtcdR::lacZ This work pRT825 pHK23::PflgM::lacZ This work pRT828 pHK23::PtcdA::lacZ Pcpr::sigD This work pRT829 pHK23::PtcdB::lacZ Pcpr::sigD This work pRT830 pHK23::PtcdR::lacZ Pcpr::sigD This work pRT831 pHK23::PflgM::lacZ Pcpr::sigD This work pRT809 pBL100::tcdR::ermB (Targetron retargeting sequence

AAAAAAGCGATG) This work

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Table S2. Primers used in this study. Primer Name Sequence (5’ to 3’) Reference rpoCqF CTAGCTGCTCCTATGTCTCACATC (11) rpoCqR CCAGTCTCTCCTGGATCAACTA (11) tcdAqF GGAGAAGTCAGTGATATTGCTCTTG This study tcdAqR CAGTGGTAGAAGATTCAACTATAGCC This study tcdBqF AAGGAATATCTAGTTACAGAAGTATTAGAGC This study tcdBqR GCAGTGTCATTTATTTGACCTCCA This study tcdRqF AGCAAGAAATAACTCAGTAGATGATT This study tcdRqR TTATTAAATCTGTTTCTCCCTCTTCA This study tcdCqF AAATAGCAAATTGTCTGATGCTGAAC This study tcdCqR TTTAGCTTCTTCAGCTTTACGTTGAT This study sigHqF TTTGCGGAGATATGTATAACAAGACAA This study sigHqR AAAGTGTTCTGTCTGACTCTTCATC This study codYqF ATTAGGAACATTGGTACTTTCAAGAT (3) codYqR TTGAACTACAGCTTTCTTTCTCATTT (3) sigDqF GAATATGCCTCTTGTAAAGAGTATAGCA (3) sigDqR TGCATCAATCAATCCAATGACTCC (3) flgMqF CAAGTGTATCAAATATGAGCGATGAA (3) flgMqR TTATCCTCGCATCTCCTCTATCATT (3) fliCqF TACAAGTTGGAGCAAGTTATGGAAC This study fliCqR GTTGTTATACCAGCTGAAGCCATTA This study fliIqF GTCTTGGAAATCCTATAGACAACTCAG This study fliIqR CATCTATTGCTCTTACACCTGTTTCC This study CD0240qF GCTAAGGGCGAATGGTTACTTATATT This study CD0240qR TTAGTCGTACTGAGGCAGAGTTAAA This study CD0241qF CCAGATAGATGTACTGTTGTGACAAAT This study CD0241qR TCAATGAAATCACCATTTGAATGAGC This study sigDCdeF CAGGATCCCTAAGGAGGCGTAGTTAATG This study sigDCdeR GACCTGCAG ATCTTATCAATCACCATCTATATAG This study motAqF CAATAGAGAGTGATGTAATGGGAATAGAAG This study motAqR CTCTAGTTCTAAGATGGACCTTATCTC This study PtcdAFB CCAGATCTCACAAAGATGGTGCATGGT This study PtcdARH CCAAGCTTAGTATTATTATTTTTGATAATAAATCCACTTC This study PtcdBFB CCAGATCTTATCTAGACAAGCTGTTAATAAGG This study PtcdBRH CCAAGCTTCTATAATATTTTTACATCTAAATGCTAAAAC This study PtcdRFB CCAGATCTCTAGTTATAACTTCAAAAAAGACTG This study PtcdRRH CCAAGCTTCTTATATTTATAATGATGATTTATTTGAAAATTTTG This study PflgMFB CCAGATCTATAAGAATGTAGATGAAAAATTAGACA This study PflgMRH CCAAGCTTAACCTCACCTCCAAAATTAC This study PcprF-XbaI CCTCTAGAAATGGTTTAGACATGGAAGTAG This study sigDR-XbaI CATCTAGAATCTTATCAATCACCATCTATATAG This study EBSuniv CGAAATTAGAAACTTGCGTTCAGTAAAC Sigma-Aldrich

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tcdRibs1 AAAAGCTTTTGCAACCCACGTCGATCGTGAAAAAAAAGCGATGGTGCGCCCAGATAGGGTG This study

tcdRebs1 CAGATTGTACAAATGTGGTGATAACAGATAAGTCGCGATGCTTAACTTACCTTTCTTTGT This study

tcdRebs2 CGCAAGTTTCTAATTTCGGTTTTTTTTCGATAGAGGAAAGTGTCT This study tcdRrev CCTCAAAAACAGACTTACTTTG This study

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Supplemental Figures

Figure S1. The C. difficile flagellar locus. The flagellar locus of C. difficile 630 spans

40.3 kilobases, from nucleotides 293002 – 333302 as shown. Previous studies suggest

that flgB through CD0272 comprise an “early stage” flagellar operon (14). The green

bent arrow upstream of flgB represents the likely promoter controlling expression of this

operon based on the recent identification of a transcriptional start site 564 bp 5’ of the

flgB open reading frame (15). Additional promoters may be present within the flgB

operon, but have not been determined experimentally. Also noted upstream of the flgB

gene is the c-di-GMP riboswitch Cd1 (pink wrench) with its transcription terminator

(black stem loop), which appears to control transcription in response to c-di-GMP (15,

16). The genes flgM through fliC were previously predicted to comprise a “late stage”

flagellar operon, and a promoter (green bent arrow) based on this prediction is shown

upstream of flgM (14). A Rho-independent transcription terminator is predicted

downstream of fliC (black stem loop, identified using the TransTermHP tool). The

CD0240-CD0244 genes do not encode flagellar structural proteins, but include two

genes annotated as encoding glycosyltransferases; one of these genes, CD0240, was

previously shown to be required for glycosylation of the flagellin FliC (17). Possible

promoters (green bent arrows) in this region are noted upstream of CD0240, following

the Rho-independent transcription terminator noted above, and upstream of CD0241,

though the promoters require experimental validation. Genes analyzed by qRT-PCR in

the current study are indicated by red boxes.

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Figure S2. The abundance of flagellar gene transcripts representing at least 3 distinct

operons are reduced in C. difficile with elevated c-di-GMP. C. difficile 630 with pDccA or

vector was grown in BHIS-Tm medium with or without 1 µg/ml nisin to early stationary

phase (OD 600nm ~ 1.0-1.2). Expression of the indicated genes was assessed by qRT-

PCR. The codY gene served as a control not regulated by c-di-GMP (3). Shown are the

means and standard deviations from at least three independent samples. The data for

each transcript were analyzed by one-way ANOVA and Dunnett’s multiple comparison

test comparing values to the average for the induced vector control. * p < 0.05.

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Figure S3. Induction of dccA expression and consequent effects on flagellar and toxin

gene expression in C. difficile grown in TY medium. C. difficile with pDccA or vector were

grown in TY-Tm medium, with or without 1 µg/ml nisin, to early stationary phase (OD

600nm ~ 2.0-2.5). The abundance of the indicated transcripts was measured by qRT-

PCR. The codY gene served as a control not regulated by c-di-GMP (3). Shown are the

means and standard deviations from five independent samples of C. difficile with vector

in the absence of nisin (light grey bars) or presence of nisin (dark grey bars), and C.

difficile with pDccA in the absence of nisin (white bars) or presence of nisin (black bars).

The data for each transcript were analyzed by one-way ANOVA and Dunnett’s multiple

comparison test comparing values to the average for the induced vector control. * p <

0.05, ** p < 0.01, *** p < 0.001. In addition to the data discussed in the main text, note

that tcdR, but not tcdC, is also repressed by expression of dccA during growth in TY

medium.

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Figure S4. Comparison of motility and flagellar gene expression between C. difficile 630

and JIR8094 strains. (A) The JIR8094 strain, a derivative of C. difficile 630, is non-

motile. The motility of C. difficile 630 and JIR8094 in tubes containing BHIS + 0.3% agar.

(B) C. difficile JIR8094 expresses several flagellar genes, tcdA, tcdB and tcdR at lower

levels than the 630 parental strain. C. difficile 630 and JIR8094 were grown in BHIS

medium to early stationary phase (OD 600nm ~ 1.0 - 1.2). The relative abundances of

the indicated flagellar and toxin gene transcripts were compared by qRT-PCR. The

means and standard deviations from at least three independent samples are shown. The

data for each transcript were analyzed by unpaired t-test. * p < 0.05, ** p < 0.01, *** p <

0.001.

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Figure S5. Verification of the tcdR mutation in C. difficile 630ΔErm. (A) The insertion of

the ermB cassette into tcdR using the Targetron system was confirmed by PCR. The

630ΔErm parental strain yields a ~300 bp product using tcdR-specific primers tcdRqF +

tcdRrev, which flank the insertion site for ermB (Lane 1). The tcdR::ermB mutant yields a

~2.3 kb product with the same primers, indicating the insertion the ~2 kb Targetron

(Lane 2). Using primers tcdRqF + EBSuniv, which is specific to the Targetron, no

product is obtained for the parent strain (Lane 3), while a product is obtained for the

tcdR::ermB mutant, indicating integration of the Targetron in tcdR (Lane 4). (B) The

tcdR::ermB mutant was further verified by assessing TcdA production by western blot.

The parent strain and tcdR::ermB mutant were grown in TY broth to late stationary

phase (48 hours growth at 37oC) to maximize detection of the toxin protein. Lysates of

these cultures were probed for TcdA by western blot. TcdA (~308 kDa) was readily

detectable in the parent strain, but not in the tcdR::ermB mutant. The CodY protein (~ 27

kDa) served as a loading control.

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Figure S6. The effect of a tcdR::ermB mutation on toxin gene expression. As part of the

experiment described in Figure 6, C. difficile 630 (light grey bars) and the C. difficile

630ΔErm tcdR::ermB derivative (dark grey bars), each carrying the vector pMC-Pcpr,

were grown in BHIS to mid-exponential phase (OD 600 nm ~ 0.6 – 0.8). Transcript levels

for tcdA and tcdB were measured by qRT-PCR. The sigD transcript was measured as a

control. The means and standard deviations are shown (n = 5). ** p < 0.01 by the Mann-

Whitney test comparing values of from the tcdR::ermB mutant to that of the 630

background for each transcript.

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