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Expanding the Repertoire of Gene Tools for Precise Manipulation of the Clostridium
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difficile Genome: Allelic Exchange Using PyrE Alleles.
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SUPPORTING INFORMATION
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Yen Kuan Ng≠ 1, Muhammad Ehsaan≠ 1, Sheryl Philip1, Mark Collery1, Clare Janoir2, Anne
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Collignon2, Stephen T. Cartman1, Nigel P. Minton1*
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≠
These authors contributed equally to this work.
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Author affiliations:
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1
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Biomolecular Sciences, University Park, University of Nottingham, Nottingham, NG7 2RD,
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United Kingdom. 2Université Paris-Sud, Faculté de Pharmacie, Département de Microbiologie,
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Unité EA 40-43, 92296 Châtenay-Malabry Cedex, France.
Clostridia Research Group, NIHR Biomedical Research Unit in GI Disease, Centre for
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*To whom correspondence should be addressed: Nigel P. Minton
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Clostridia Research Group, Centre for Biomolecular Sciences, University Park, University of
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Nottingham,
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Nottingham, NG7 2RD, United Kingdom
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Phone: +44 (0)115 84 67458
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Fax: +44 (0)115 82 32120
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(a)
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WT
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ter
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wa
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sm
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pla
MW
id
700 bp
600 bp
(b)
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Figure S1. PCR screening of ACE generated pyrE mutant and wildtype strains in C. difficile strains
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630∆erm and C. difficile R20291. (a) five random FOAR, pyrE mutants (lanes 1-5) made in
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R20291 using pMTL-YN18, and; (b) five random pyrE repaired clones (lanes 1-5) from R20291
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made using pMTL-YN2. MW is a 2-Log DNA Ladder (NEB) molecular weight marker, plasmid is
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a pMTL-YN18 DNA control, WT is a wild-type C. difficile DNA control, and ∆pyrE is a mutants
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control taken from the strain in lane 1 of (a). The primers used, and expected size of the DNA
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fragments generated were: (a) primers pyrE-F2/pyrE-R2 and a 565 bp DNA fragment, and; (b)
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primers cdi630-pyrD-SF1/pyrE-R2 and a 750 bp DNA fragment.
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FIGURE 10a
A
3, 000 bp
2, 000 bp
1, 500 bp
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MW
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WT
wa
MW
ter
1, 000 bp
B
4, 000 bp
3, 000 bp
2, 000 bp
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WT
wa
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ter
1, 500 bp
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3, 000 bp
2, 000 bp
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WT
wa
MW
ter
1, 500 bp
1, 000 bp
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Figure S2. PCR screening of double crossover candidate clones of C. difficile 630Δerm
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for spo0A, cwp84 and mtlD. (A) PCR screening of four (1-4) putative spo0A in-frame deletion
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clones using primers spo0A-YN-F2 and spo0A-YN-R2. The desired mutant should give a DNA
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product of 1,845 bp in size (clone 2), while the wild type allele (clones 1, 3 and 4) generates a
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product of 2,331 bp in size. (B) PCR screening of three (1-3) putative cwp84 in-frame deletion
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clones using primers cwp84-F3 and cwp84-R4. A double crossover mutant (clone 3) should give
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a PCR product of 1,418 bp in size, as opposed to the 2,636 bp fragment predicted for the wild
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type allele (clones 1 and 2). (C) PCR screening of three (1-3) putative in-frame deletions of mtlD
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using primers mtlD-F3 and mtlD-R3. The in-frame deletion mutant is predicted to yield a PCR
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DNA product 1,418 bp in size (clones 1 and 3), whereas the wild-type allele (clone 2) gives a
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DNA product of 2,582 bp in size. PCR product from wild-type or double-crossover wild-type
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revertants. MW is a 2-Log DNA Ladder (NEB) molecular weight marker and WT is a wild-type C.
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difficile DNA control.
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Figure S3. PCR screening of double crossover candidate clones of C. difficile R20291 for
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spo0A and cwp84. (A) PCR screening of three (1-3) putative spo0A in-frame deletion clones
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using primers spo0A-YN-F2 and spo0A-YN-R2. The desired mutant should give a DNA product
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of 1,845 bp in size (clones 2 and 3), while the wild type allele (clone1) generates a product of
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2,331 bp in size. (B) PCR screening of four (1-4) putative cwp84 in-frame deletion clones using
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primers cwp84-F3 and cwp84-R4. Double crossover mutants (clones 1, 2 and 4) should give a
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PCR product of 1,418 bp in size, compared to the 2,636 bp fragment predicted for the wild type
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allele (clone 3). MW is a 2-Log DNA Ladder (NEB) molecular weight marker and WT is a wild-
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type C. difficile DNA control.
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Figure S4. Comparison of colony morphology of C. difficile 630Δerm strains. C. difficile
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630Δerm (A) 630ΔermΔcwp84 mutant (B), and 630ΔermΔcwp84-complemented (C) were
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streaked onto anaerobic blood agar plate and incubated overnight to observe colony
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morphology. Colonies of C. difficile 630Δerm (A) and 630ΔermΔ cwp84-complemented (C) are
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indistinguishable and showed irregular edged colonies compared to C. difficile 630Δerm cwp84
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mutant (B) showed more rounded colonies and are noticeably smaller due to its slower growth
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rate.
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Figure S5. Complementation of spo0A mutants of 630Δerm∆pyrE and R20291∆pyrE. The
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sporulation phenotype of the mutants and complemented mutants were compared to the
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wildtype strains by assaying colony forming units (CFU) on BHIS supplemented with 0.1% [w/v]
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sodium taurocholate before and after heat shock (65°C for 30 min) following growth of each
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strain in BHIS media for 120 h. Assayed strains are: 630∆erm∆pyrE∆spo0A; 630∆erm*(spo0A);
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630∆erm; R20291∆pyrE∆spo0A; R20291*(spo0A), and; R20291, where * indicates the spo0A-
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complemented strain (spo0A) which has had a copy of the spo0A gene inserted into the
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chromosome, concomitant with the correction of the pyrE allele back to wildtype. The detection
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limit for the assay was 50 CFU/ml. All experiments were undertaken in triplicate. Phase contrast
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microscopy confirmed that phase-bright spores were absent in the mutant cultures but present
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in both the complemented and wildtype strains.
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A
1.8
1.6
1.4
1.2
630 Δerm
630 Δerm ΔmtlD
OD600
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0.8
630 Δerm ΔmtlD complemented
630 Δerm ΔmtlD overexpressed
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hours
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B
630 Δerm
630 Δerm ΔmtlD
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630 Δerm ΔmtlD complemented
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630 Δerm ΔmtlD overexpressed
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pH
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Fig. S6. Growth of C. difficile 630Δerm strains with mannitol as the sole carbon source..
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(B) The growth of ∆mtlD was limited in mannitol broth, while growth of the ∆mtlD complemented
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and mtlD overexpressed strains were restored to wildtype levels. (C) The pH of the media broth
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showed a dip in pH caused by the fermentation of mannitol for the wildtype, ∆mtlD
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complemented and ∆mtlD overexpressed strains, which correlate to their growth. The 630 ∆erm
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∆mtlD mutant strain grew very weakly in mannitol broth, which was reflected in the sustained pH
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levels of the media. Experiments were undertaken in triplicate.
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