Supp lement ary mate rial
Ident ific ati on o f t he unw inding regio n in the Clostrid ioides difficile
chromosomal origi n of replication
Ana M. Oliveira Paiva 1,2 , Erika van Eijk 1 , A n nemieke H. Friggen 1 , Christoph Weigel 3 , Wiep Klaa s
Smits 1,2 *

1. S uppleme ntary Data

Patte rn search :
NTA TCCACA
TNA TCCACA
TTA NCCACA
TTATCNAC A
TTA TCCNCA
TTATCC ANA
TTA TCCACN
TTN TCCACA
TGTGGAT AN
TGTGGAT NA
TGTGGNT AA
TGTNGATAA
TGNGGAT AA
TNTGGATAA
NGTGGAT AA
TGTGGAN AA
TTWTNCAC A
TGTGNA WAA
NTW TNCA CA
TGTGNA WAN
TNW TNCA CA
TGTGNA WNA
TTW NNCA CA
TGTGNN WAA
TTWTNNAC A
TGTNNAWAA
TTWTNCNCA
TGNGN AWAA
TTWTNCA NA
TNTGNAWAA
TTWTNCAC N
NGTGN AWAA

2. S uppleme ntary Fig ures

Figu re S 1 . Alignm ent of d omain IV of the C. dif ficile and Therm otoga mariti ma DnaA protein .
Res idues a re colo red a cc ording to s equ ence i denti ty co nse rvati on us ing blue sha ding ( dar k
blue more cons erved), a s analysed in JalView , as for Figure 1. Residues i nvolved in specif ic
conta cts w ith the 9 - mer DnaA box sequ ence a re indic ate d in orang e. It i s c lear tha t the
majo rity o f these resid ues are not c ons erv ed b etween the tw o spec ies , exc ept C. difficile
R370/ T. maritima R36 6.

Figu re S 2. Expres sion and p urifica ti o n of C. d iffici le DnaA - 6xH is pro tein. A) E. c oli expressing
DnaA - 6xHis cells were induced with 1 mM IPTG. Optical density - normali zed samples before
induc tion (T 0), a fter 1 hour of i nduc tion ( T1) a nd 3 hour s of i nduc tion (T3) were re solv ed by
12% SDS - P AGE a nd i mmuno bl otte d with a nti - his anti body . I nduc ed DnaA is obs erve d wit h the
approximate molecular weight of 51 kDa (red arrow) . Possible breakdown product is observed
(blue arrow). B) . Sample s of Dn aA - 6xHis HisTra p pur ific ati on fr om the el utio n fracti on 2 at
bindi ng b uffer wit h diffe rent i midazol e co ncentr ations ( 20, 60, 100, 300 and 5 00 mM) w ere
sep ar ated b y 12% SDS – PAGE a n d s t ained w ith Co omassie brilli ant bl ue. DnaA - 6xHis i s
observed with an approximate m olecular weig ht of 51 k Da (red arrow ), and elute d in Binding
buffer suppl eme nted w ith > 300 mM i mida zole. C ) Confi rma tio n of size - exclusion fraction
conta ini ng the C. difficile DnaA - 6xHis and fur ther use d for anal ysi s aft er pr otein pu rific ation
resolved by 12% SDS - PA GE ( Cooma ssi e s tai ning) and immuno blotte d wi th a nti - his antibody.
DnaA - 6xHis is observed w it h the approximate molecular weight o f ~ 51 kDa (red arrow).
Possible minor b reakdown products are observ ed (gree n as ter isk).

Figu re S 3 . P1 nucleas e ass ay of the indiv idual C. diff icile or iC r egions . A ) Repr ese nta tio n of
the oriC regio ns pres ent i n the use d vect ors for P1 nucleas e ass ay, oriC1ori C2 (pAP205) , oriC1 -
(pAP83) a nd oriC2 (pA P76) - contai ning v ectors . The pr edict ed oriC regions (dotted lines) and
incl uded g enes are repre sented, rpmH (b lue), dnaA (orange), and dnaN (gree n). P 1 nucleas e
assay of pAP8 3 ( oriC1 , upper panel) and pAP76 ( oriC2 , lower panel). Dige st ion of the v ector
wi th the re stric tion enzy mes B glII ( left panel) or N otI (r igh t panel) . Dig est ion of the v ector s
wi th the re s tricti on enzy mes ( lanes 1 - 3) . Trea tme nt of the frag me nts w ith P1 nucle as e only
(la ne 2) and i ncuba ted w ith 0.1 4 µM of C. difficile D naA - 6xHis p rote i n (lan e 3). Higher D naA -
6xHis w ere tested wit h same profile ( data no t show n). The DNA fragments w ere separated in
a 1% agarose gel a nd analyzed with et hidium bromide s taining. Spo ntan eous unw inding is
obser ve d and n o Dna A - depen dent unwin ding is d etec ted.

Figu re S 4 . Quantif icatio n of the P 1 - indepe nden t ba nds. Da ta prese nted here a re
com pleme ntary to that of F igur e 4C a nd 4 D in the mai n body of the ma nuscr ipt.
Quantification was p erformed u sing Imag eJ, and s ignals were normalized to the total signal
in a la ne . A . Resul ts for P1/ BglII digest ed vector. S hown is the signal (black circles) for the
upper ban d of the gel ( Fi gure 4B , uppe r pa nel). B . R esults for the P1/ No tI digeste d vec tor .
Show n is the q uanti ficati on of the sig nal of the upper ( black circ les) a nd lower (ope n circle s)
bands of the g el (Figure 4 B, lower pa nel). Error bars indicate the st andard deviation of the
mean of n= 3 in depen dent exp erime nts.

Figu re S 5 . SIDD a nalysis diffe rent clos tridia. Anal yis of 2.0 kb fragme nts comprising oriC1
and oriC2 in C. difficile R 20291, C. bot ulinum A Hall, C. sor delli AM370, C. ace tobut yli cum
DSM 1731, C. perf ringens str.13, C. tet ani E88 (se e Tab l e 1 in the m ain body of t he
manuscript ). Nuc leoti de posi tioni ng is i ndicat ed. Predic ted fr ee e nergies G( x) for d uplex
desta biliza tio n at a supe rhel ica l dens ity of σ = - 0.0 6 (green) or σ = - 0.04 (r ed).

Fig. S 6. Comparis on of the B. subt ilis and C. dif ficile ori C2 . Repr esenta tion of the oriC2 re gio n
(interg enic r egion betw een dnaA and dnaN ) of B. subtilis and C. difficile chr omoso m e. The dnaA and
dnaN genes a re rep res ented by o ran ge and green arrows, re spectively. The DUE i s repre sented by a
grey circle. DnaA - trio sequenc es are show n in light blue boxes. D naA - boxes are indicat ed by pink
box es and orient ation on t he leading (right) and laggin g strand (left ) are show n. DnaA boxe s are
numbe red accor ding to the B. subtilis nomencl ature (Richa rdso n, 2019 ), w ith numbe rs in blu e ( no
mismatch from th e TTATCCACA seque nce , red ( 1 mismatch), black ( 2 mismatches) or y e l lo w ( 3
mismatches). See Material and Methods f or detailed information. Align m ent of t he represented
chrom osomal regions i s ba sed o n the loca tion of t he DnaA - trio.

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