Mic oo ganisms 2022, 10, 516. h ps://doi.o g/10.3390/mic oo ganisms10030516 www.mdpi.com/jou nal/mic oo ganisms
A icle
C yp ic P ophages Con ibu ion o Campylobac e jejuni and
Campylobac e coli In og ession
Luís Tanoei o
1
, Mónica Oleas o
2
, Alexand a Nunes
3
, And eia T. Ma ques
1
, Síl ia Vaz Dua e
4
,
João Paulo Gomes
3
, An ónio Ped o Al es Ma os
5
, Jo ge M. B. Ví o
1
and Filipa F. Vale
1,
*
1
Pa hogen Genome Bioin o ma ics and Compu a ional Biology, Resea ch Ins i u e o Medicines
(iMed-ULisboa), Facul y o Pha macy, Uni e sidade de Lisboa, 1649-003 Lisboa, Po ugal;
[email p o ec ed] (L.T.); and eia. .ma [email protected] (A.T.M.); j i o @ .ulisboa.p (J.M.B.V.)
2
Na ional Re e ence Labo a o y o Gas oin es inal In ec ions, Depa men o In ec ious Diseases, Na ional
Ins i u e o Heal h D . Rica do Jo ge, 1600-609 Lisboa, Po ugal; monica.oleas [email protected]
3
Bioin o ma ics Uni , Depa men o In ec ious Diseases, Na ional Ins i u e o Heal h D Rica do Jo ge,
1600-609 Lisboa, Po ugal; alexand [email protected] (A.N.);
[email protected] (J.P.G.)
4
Inno a ion and Technology Uni , Depa men o Human Gene ics, Na ional Ins i u e o Heal h D Rica do
Jo ge, 1600-609 Lisboa, Po ugal; sil ia.du[email p o ec ed]saude.p
5
Cen o de In es igação In e disciplina Egas Moniz (CiiEM), Coope a i a de Ensino Supe io Egas Moniz,
Quin a da G anja, 2829-511 Capa ica, Po ugal; apam[email p o ec ed]
* Co espondence: ale. [email protected] o . ale@ .ulisboa.p
Abs ac : Campylobac e coli and C. jejuni, he causing agen s o campylobac e iosis, a e desc ibed o
be unde going in og ession e en s, i.e., he ans e ence o gene ic ma e ial be ween di e en spe-
cies, wi h some isola es sha ing almos a qua e o i s genome. The pa icipa ion o phages in in-
og ession e en s and consequen impac on hos ecology and e olu ion emain elusi e. Th ee dis-
inc p ophages, named C. jejuni in eg a ed elemen s 1, 2, and 4 (CJIE1, CJIE2, and CJIE4), a e de-
sc ibed in C. jejuni. He e, we iden i ied wo un epo ed p ophages, Campylobac e coli in eg a ed
elemen s 1 and 2 (CCIE1 and CCIE2 p ophages), which a e C. coli homologues o CJIE1 and CJIE2,
espec i ely. No induc ion was achie ed o bo h p ophages. Con e sely, induc ion assays on CJIE1
and CJIE2 poin owa ds he inducibili y o hese p ophages. CCIE2-, CJIE1-, and CJIE4-like p o-
phages we e iden i ied in a Campylobac e spp. popula ion o 840 genomes, and phylogene ic anal-
ysis e ealed clus e ing in h ee majo g oups: CJIE1-CCIE1, CJIE2-CCIE2, and CJIE4, clea ly seg-
ega ing p ophages om C. jejuni and C. coli, bu no om human- and nonhuman-de i ed isola es,
co obo a ing he lowing be ween animals and humans in he ag icul u al con ex . Punc ual bac-
e iophage hos -jumps we e obse ed in he con ex o C. jejuni and C. coli, and al hough andom
chance canno be ully disca ded, hese obse a ions seem o implica e p ophages in e olu iona y
in og ession e en s ha a e modula ing he hyb idiza ion o C. jejuni and C. coli species.
Keywo ds: bac e iophage; in og ession; Campylobac e ; hos ange
1. In oduc ion
Campylobac e species ela ed o campylobac e iosis cases shows ha 83.9% o he
iden i ied bac e ia we e C. jejuni, 10.3% we e C. coli, while he emaining a e o he Cam-
pylobac e species [1]. Thus, he he mo ole an C. jejuni and C. coli a e he majo sou ces
o human campylobac e iosis [2–4], mos ly a ibu ed o poul y mea handling and con-
sump ion [5,6]. These wo species a e hough o ha e di e ged o e 6500 yea s ago du -
ing he Neoli hic e olu ion, coinciding wi h animal domes ica ion and changes in ag i-
cul u e p ac ices, while C. coli popula ion di e ged in o h ee dis inc clades (clades 1, 2,
and 3) by abou 1700–1000 yea s ago [4,7]. The appea ance o clonal complexes in he
popula ion occu ed way a e he species di e gence [4].
Ci a ion: Tanoei o, L.; Oleas o, M.;
Nunes, A.; Ma ques, A.T.;
Dua e, S.V.; Gomes, J.P.;
Ma os, A.P.A.; Ví o , J.M.B.;
Vale, F.F. C yp ic P ophages
Con ibu ion o Campylobac e jejuni
and Campylobac e coli In og ession.
M
ic oo ganisms 2022, 10, 516.
h ps://doi.o g/10.3390/mic oo ganis
ms10030516
Academic Edi o : Igo V. Babkin
Recei ed: 12 Janua y 2022
Accep ed: 23 Feb ua y 2022
Published: 26 Feb ua y 2022
Publishe ’s No e: MDPI s ays neu-
al wi h ega d o ju isdic ional
claims in published maps and ins i u-
ional a ilia ions.
Copy igh : © 2022 by he au ho s. Li-
censee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and con-
di ions o he C ea i e Commons A -
ibu ion (CC BY) license (h ps://c e-
a i ecommons.o g/licenses/by/4.0/).
Mic oo ganisms 2022, 10, 516 2 o 17
An in e es ing phenomenon occu ing in he na u al compe en Campylobac e spp.
[8] is he ans e ence o gene ic ma e ial be ween di e en species, he so-called in o-
g ession. C. jejuni and C. coli om clade 1, which a e ca. 12% di e gen a nucleo ide se-
quence le el (as much as humans a e om ma mose ) [4], equen ly exchange gene ic
ma e ial h ough ho izon al gene ans e . The ex ensi e in og ession, mo e common in
C. jejuni- o-C. coli di ec ion, led o he eplacemen o ≈10% and ≈23% o C. coli co e genome
wi h C. jejuni DNA, in ST-828 and ST-1150 clonal complexes, espec i ely. These wo
clonal complexes in C. coli clade 1 a ose by C. jejuni DNA accumula ion and cons i u e he
as majo i y o yped isola es om his clade [9]. The in og ession le els in he opposi e
di ec ion o in ol ing C. coli isola es om clades 2 and 3 a e a less common [9] in he
case o he las wo, p obably due o ecological ba ie s [4]. Thus, hese exchanges may
a i e om ecen changes in he ecologic niche d ew by human ac i i y [10]. In og es-
sion ep esen s a sou ce o adap i e alleles being d i en by niche o e lap be ween ecip-
ien and dono species, which may esul in hyb id specia ion [11] con ibu ing o he
o igin o a new species, o e en despecia ion [10] esul ing in he usion o wo species.
The i s p ophages wi hin Campylobac e genome we e desc ibed yea s a e he se-
quencing o C. jejuni NCTC 11,168 [12], which ha bo s no p ophages. Th ee dis inc C.
jejuni in eg a ed elemen s (CJIE) iden i ied in C. jejuni s ain RM1221 assume majo im-
po ance: CJIE1, CJIE2, and CJIE4. CJIE1 is a Campylobac e Mu-like phage (also known as
CMLP1) [12], appa en ly inducible wi h mi omycin C, ha encodes se e al p o eins wi h
simila i ies o bac e iophage Mu and o he Mu-like p ophages. CJIE2 and CJIE4 a e simi-
la p ophages and encode ew s uc u al p o eins. In eg a i e elemen CJIE3 has been de-
sc ibed bu poin ed ou as an in eg a i e plasmid [12]. Recen ly, a new in eg a ed elemen
called CJIE5 p ophage has been p oposed [13]. In ec ious CJIE1 and CJIE4 phage pa icles
ha e been desc ibed o be di icul o ob ain [14–16], suppo ing CJIE-like p ophages o be
incomple e o emnan p ophages. Howe e , CJIE p ophages ha e been epo ed o ha e
impac on Campylobac e spp. ea u es. Indeed, CJIE1-ca ying isola es showed signi i-
can ly inc eased adhe ence and in asion when compa ed o nonca ie s, in a mobili y-
and g ow h-independen manne [17]; p ophage-encoded DNases we e epo ed o in-
hibi na u al ans o ma ion in C. jejuni isola es ca ying CJIE1, CJIE2, and CJIE4 p o-
phages [18,19]; and i was also shown ha he ca iage o CJIE1 p ophage a ec s p o ein
exp ession, including i ulence-associa ed p o eins [16].
Whe he C. coli genomes p esen p ophage homologues o C. jejuni p ophages o i
Campylobac e p ophages ha e he abili y o in ec bo h C. jejuni and C. coli emains o be
de e mined and a e he aims o he p esen s udy. To add ess hese poin s, we ha e ana-
lyzed 177 Campylobac e p ophage sequences among 840 genomes, o which 22 C. jejuni
and 82 C. coli a e newly sequenced genomes isola ed om human and nonhuman hos s,
and 692 C. jejuni and 44 C. coli a e genomes a ailable in public da abases.
2. Ma e ials and Me hods
2.1. Campylobac e Genomes
A o al o 104 genomes om non ela ed Po uguese Campylobac e spp. isola es, 22 C.
jejuni (15 clinical and 7 poul y) and 82 C. coli (43 clinical and 39 poul y) (Table S1) we e
selec ed o p ophage sc eening. Whole genome sequencing (WGS) and de no o assembly
we e pe o med as p e iously desc ibed [20]. Raw sequence eads o six s ains ( ou C.
coli and wo C. jejuni) ep esen a i e o he p ophages di e si y we e deposi ed in he Eu-
opean Nucleo ide A chi e (ENA) unde he s udy accession numbe s PRJEB46733 and
PRJEB46750, espec i ely. Fo compa a i e pu poses, p ophage sc eening was also e al-
ua ed on 692 C. jejuni and 44 C. coli genomes e ie ed om PATRIC [20].
Mic oo ganisms 2022, 10, 516 3 o 17
2.2. P ophage Sc eening Using Bioin o ma ic Tools
PHASTER [21] and P ophage Hun e Tool (PHT) [22] we e used o iden i ica ion o
po en ial p ophagic egions in he newly sequenced C. coli and C. jejuni genomes, consid-
e ing only egions p edic ed as in ac (by PHASTER) o ac i e (by PHT). An addi ional
25,000 bp minimum egion leng h was es ablished, as o e 90% (66/72) o Campylobac e
spp. phage genomes a ailable a PATRIC a e >25,000 bp. The inse ion si es we e de e -
mined using he e e ence genome C. coli 15-537,560 (GenBank Accession: CP006702) [23]
as p ophageless empla e. P ophages we e anno a ed using RAST [24], and u he se-
quence analysis was done using BLAST [25], while s uc u al homology analyses we e
pe o med using Phy e2 [26].
2.3. P ophage Induc ion
P ophage induc ion was pe o med using 2 µg/mL mi omycin C [27,28] o 0.15% so-
dium deoxychola e [15], as desc ibed elsewhe e [15,27,28] o he C. jejuni s ains Cj7 and
Cj18 (ha bo ing CJIE1- (MZ667637) and CJIE2-like (MZ667636) p ophages, espec i ely),
and C. coli s ains Cc84 and Cc11 (ha bo ing CCIE1- (MZ667638) and CCIE2-like
(MZ667639) newly iden i ied p ophages, espec i ely). Phage-induced lysis was es ed in
bac e ial lawns o po en ially indica o s ains C. jejuni Cj11 and C. coli Cc88 (p edic ed o
ha bo no p ophage).
Phage DNA om concen a ed phage pa icles was ex ac ed wi h QIAp ep Spin
Minip ep Ki (Qiagen, Ge man own, MD, USA), ollowing manu ac u e ’s ins uc ions
o la ge plasmid (>10 kb). To ensu e comple e bac e ial DNA elimina ion, sequen ial di-
ges ion wi h Exonuclease I (E. coli) (New England Biolabs, Ipswich, MA, USA) and
Lambda Exonuclease (New England Biolabs) was pe o med as desc ibed elsewhe e [29].
P esence o phagic and bac e ial DNA was es ed by PCR a ge ing CJIE1-CCIE1 mo pho-
genesis p o ein gene, CJIE2-CCIE2 e minase gene, and Campylobac e spp. glu amine syn-
he ase gene (Table S2). Phage pa icles we e obse ed by ansmission elec on mic os-
copy (JEOL 100SX) a e nega i e s aining, as p e iously desc ibed [29].
2.4. Phylogene ic Analysis
A e MAFFT e sion 7 [30] alignmen , a maximum-likelihood phylogene ic ee us-
ing Jukes–Can o model o nucleo ide e olu ion [31] was cons uc ed wi h Fas T ee 2.1
[32]. P ophages CJIE1-1 o 1-4 (HM141978, HM192820, HM581889, and HM543163) and
CJIE4-1 o 4-5 (KF751793, KF751794, KF751795, KF751796, and KF751797), as well as
CCIE2 p ophage (MZ667634), we e included as model p ophages ep esen a i e o each
sub ee. The CJIE2 and CJIE3 egions o C. jejuni RM1221 (NC_003912.7) we e ex ac ed
and included. En e obac e ia phage Mu (NC_000929) [33] was included as ou g oup.
T ees we e isualized using In e ac i e T ee O Li e (iTOL) 4 [34].
2.5. Tes ing In og ession Using ABBA-BABA S a is ics
ABBA-BABA s a is ics, o D s a is ics, was used o es o in og ession using single
nucleo ide polymo phism (SNP) da a [35–38], allowing o de e mine i in og ession has
occu ed, and be ween which axa, based on expec a ions o he equencies o di e en
gene ee opologies [39]. An excess o a SNP pa e n is indica i e o in og ession, i.e.,
gene low be ween wo o he axa [37]. The in og ession was es ed o CJIE4 and o
CCIE2 and CJIE2 p ophages, since hese we e he cases whe e p ophage spillo e be ween
C. jejuni and C. coli species was de ec ed by phylogene ic analysis. A mul iple sequence
alignmen using MAFFT [30] was pe o med o he CJIE4 p ophages and ano he o
CCIE2 and CJIE2 p ophages, using in bo h cases En e obac e ia phage Mu (NC_000929)
[33] as ou g oup. SNPs we e ex ac ed om mul iple sequence alignmen s using SNP-
si es [40]. Using an R sc ip , he allele equencies a each SNP we e de e mined, ollowed
by D s a is ic and block jackkni e me hod o es o a signi ican de ia ion om he null
Mic oo ganisms 2022, 10, 516 4 o 17
hypo hesis D = 0 [35,36]. The admix u e p opo ion was de e mined using d s a is ic
[37,38,41].
2.6. P ophage Nuclease Sc eening
The iden i ied p ophages we e sc eened o nucleases using CJIE1-encoded endonu-
clease dns (locus ag: CJE0256 in C. jejuni RM1221), o he CJIE2- and CJIE4-encoded endo-
nuclease nucA (locus ag: CJE0566 in C. jejuni RM1221 and locus ag: 01-1512_00025 in Cam-
pylobac e phage CJIE4-5, espec i ely) [18,19]. In ei he case, genes wi h co e age >90%
we e conside ed as comple e genes, while genes wi h co e age 50–90% we e conside ed
as pa ial. Lowe co e ages we e epo ed as no de ec ed.
3. Resul s
3.1. Iden i ica ion o P ophages
Wi hin he 22 C. jejuni and 82 C. coli newly sequenced genomes, a o al o 402 p o-
phage egions we e p edic ed (123 PHASTER-iden i ied and 279 PHT-iden i ied, mean
leng h o 18,340.47 ± 9773.85 bp, anging om 4543 o 50,845 bp—da a no shown). Con-
side ing only p edic ed p ophages wi hou homology wi h plasmids, la ge han 25,000
bp, p esen ing s uc u al p o eins, and classi ied as in ac (by PHASTER) o ac i e (by
PHT) educes he lis o nine by PHASTER (2 in C. jejuni and 7 in C. coli) and 29 by PHT
(4 in C. jejuni and 25 in C. coli) (Table 1). PHASTER and PHT clea ly iden i ied h ee g oups
o p ophages wi h homology wi h CJIE1, CJIE2, and CJIE4. The nine PHASTER-iden i ied
p ophages e idenced homology wi h CJIE1 (a e age pe cen iden i y o 81.1% o C. coli
and 93.1% o C. jejuni p edic ed p ophages). Among he PHT-p edic ed p ophages, 28
had homology wi h CJIE2 (a e age pe cen iden i y o 54.5% o C. coli and 52.8% o C.
jejuni p edic ed p ophages), and one wi h CJIE4 (pe cen iden i y o 91.7% o a C. coli
p ophage). These obse a ions oge he wi h he phylogene ic analysis (see below) pin-
poin he exis ence o wo no el p ophages in C. coli ha o hei simila i y wi h C. jejuni
p ophages we e named Campylobac e coli in eg a ed elemen 1 (CCIE1) and Campylobac e
coli in eg a ed elemen 2 (CCIE2). Al hough CJIE1-like p ophages ha e been conside ably
epo ed in C. coli genomes [42], he majo i y (i no all) o hem may be, in ac , CCIE1
p ophages. In e es ingly, CJIE2 p ophages we e only epo ed in C. jejuni isola es so a
[12,42–47], whe eas, in con as , CCIE2 p ophages we e ound in bo h C. coli and C. jejuni
genomes. CCIE1 and CJIE1 a e e y simila o each o he (≈80% sequence iden i y), while
CCIE2 and CJIE2 do no show such simila i y (≈50% sequence iden i y). Signi ican dele-
ions and ea angemen s in CJIE2-like p ophages we e desc ibed [44,45], po en ially ex-
plaining he low co e age o homologous egions when compa ing CJIE2 and CCIE2 p o-
phages. No ably, CJIE1-CCIE1 and CJIE2-CCIE2 display se e al genes sha ed in block
wi h same o ganiza ion (da a no shown). Bo h CCIE1 and CCIE2 showed genome leng h
o 38,556 bp and 36,356 bp, espec i ely, consis en wi h CJIE1 (34403 bp) and CJIE2 (40268
bp).
Table 1. Numbe o p edic ed p ophage egions wi hin newly sequenced C. coli and C. jejuni ge-
nomes using PHASTER [21] and P ophage Hun e [22].
De ec ed P ophage Sequences * C. jejuni (n = 22) C. coli (n = 82) To al (n = 104)
Genomes wi hou p ophage sequences 1 5 6
PHASTER ( o al) 39 84 123
PHASTER (in ac ) 2 7 9
P ophage Hun e Tool ( o al) 84 195 279
P ophage Hun e Tool (ac i e) 4 25 29
* Pa en hesis: o al numbe o phages and numbe o phages p edic ed o be comple e by PHASTER
(so wa e classi ica ion in ac ) and P ophage Hun e (so wa e classi ica ion ac i e).
Mic oo ganisms 2022, 10, 516 5 o 17
3.2. Cha ac e iza ion o he New CCIE1 and CCIE2 P ophages
A mo e de ailed analysis was pe o med on he CCIE1 egion wi hin C. coli Cc63-H-
18 genome, and on he CCIE2 egion wi hin C. coli Cco1598-H-13 genome (Figu e 1, Tables
2 and S3). Mos p edic ed CDS in bo h CCIE1 e CCIE2 we e ma ched ei he by sequence
o s uc u e wi h phage genes (Table S3). Se e al hypo he ical p o eins we e anno a ed as
a esul o he lack o knowledge su ounding Campylobac e spp. phages. A bac e ial pu-
a i e NADH-ubiquinone oxido educ ase, loca ed a he 3′ edge o CCIE2 genome, was
iden i ied, po en ially ep esen ing a wa e ma k om a pas phage ansduc ion e en .
Mo e speci ically, he pe iphe al loca ion o he pu a i e bac e ial gene in he p ophage
genome poin s o specialized ansduc ion, in which he bac e iophage packages i s ge-
nome wi h lanking bac e ial DNA aken du ing ch omosomal excision [48].
Rega ding s uc u al analysis, CCIE1 seems a nea -comple e p ophage lacking only
one essen ial s uc u al p o ein, possibly explaining he gene al ailu e o induc ion a -
emp s o i s close homologue CJIE1 p ophage [15,49], while no objec ing a case o appa -
en CJIE1 induc ion success [12]. Howe e , i should no be uled ou ha induc ion ailu e
may be due o ine icien expe imen al condi ions. On he o he hand, CCIE2 was p e-
dic ed o ha e se e al s uc u al p o eins missing, simila o i s homologue CJIE2 [12]. The
lack o s uc u al p o eins is a common ea u e o c yp ic p ophages [50], which sugges s
ha CCIE2 is possibly incomple e.
Figu e 1. Genome anno a ion o he newly iden i ied CCIE1 (a) and CCIE2 (b) p ophages. The ge-
nome o bo h CCIE1 and CCIE2 p ophages and hei anno a ed CDS a e ep esen ed. Hypo he ical
p o eins o which no anno a ion upda e was possible a e ep esen ed in yellow. Regula ion p o-
eins and genome p ocessing p o eins a e depic ed, espec i ely, in blue and g een, while lysis- e-
la ed p o eins and s uc u al p o eins a e highligh ed in ed and g ey, espec i ely. Pa ial dns nu-
clease ound in CCIE1 is shown in lilac. Fo space simpli ica ion, he linea p ophage egion is ep-
esen ed as a ci cula genome, wi h s a ( i s esidue) and end (las esidue) highligh ed a 0′ posi-
ion o each ep esen a ion. The igu e was ob ained using Geneious P ime 2020.1.1. Fu he de-
sc ip ion o he anno a ed egions is a ailable on Table S3.

Mic oo ganisms 2022, 10, 516 6 o 17
Table 2. Gene al cha ac e iza ion o he newly iden i ied CCIE1 and CCIE2 p ophages.
Cha ac e is ics CCIE1 P ophage CCIE2 P ophage
Genome leng h 38,556 bp 36,356 bp
GC con en 30,20% 28,50%
GenBank Accession No. MZ667635 MZ667634
Closes homologue CJIE1-2 (HM192820.1) [51] CJIE2 [12]
Co e age wi h he closes homologue 79% 54%
Iden i y wi h he closes homologue 96.33% 93.85%
Hos s ain 1 C. coli Cc63-H-18 C. coli Cco1598-H-13
Inse ion si e 2 5′ Bis-ABC ATPase YbiT (N149_0417) RNA-Leu-GAG (N149_0910)
3′ pu a i e lipop o ein (N149_01930) pu a i e NTPase (N149_01865)
Numbe o CDS 3 59 54
Main phage genes de ec ed 3
In eg ase, dns nuclease, endolysin,
holin, me hylase, e minase, se e al
phage s uc u al and egula ion
p o eins.
In eg ase, endolysin, ecombinase/ex-
onuclease, me hylase, esol ase, e -
minase, se e al phage s uc u al and
egula ion p o eins.
VIRFAM analysis
Almos comple e Mu-like Myo i i-
dae phage (head closu e p o ein
missing)
Incomple e phage (se e al s uc u al
p o eins missing)
1 Raw sequence eads we e deposi ed in he Eu opean Nucleo ide A chi e (ENA) unde he s udy
accession numbe PRJEB46733; 2 locus ag in he genome o he e e ence s ain C. coli 15-537560;
3 u he de ails on Table S3.
3.3. P ophage Induc ion Assays
Despi e being epo ed as mi omycin C-inducible [12], o ou knowledge CJIE1 in-
duc ion was no desc ibed in de ail and he e a e a couple epo s o induc ion ailu e
[15,16]. CJIE2, on he o he hand, was epo ed as incomple e; hus, likely no inducible
[12]. Fo he CJIE1-like p ophage, PCR a e exonuclease ea men s ende ed phagic gene
ampli ica ion (Figu e 2A, lanes 6, 12, and 18). Simila esul s we e ob ained o he CJIE2-
like p ophage (da a no shown), sugges ing ha bo h C. jejuni-ha bo ed p ophages a e
inducible. The de ec ion o ci cula phagic genomes o no -induced bac e ia (noninduced
con ol), poin s owa ds a basal spon aneous elease o phage pa icles. Fo he CCIE1-
like p ophage, PCR a e exonuclease ea men s ende ed no phagic gene ampli ica ion
(Figu e 2B, lanes 6, 12, and 18). Simila esul s we e ob ained o he CCIE2-like p ophage
(da a no shown), sugges ing ha CCIE1 and CCIE2 we e no inducible, hus ende ing
no PCR ampli ica ion. Despi e hese obse a ions, no phage-induced lysis was obse ed
upon applica ion o PCR posi i e phage p ecipi a es on he po en ially indica o s ains
(da a no shown).
Mic oo ganisms 2022, 10, 516 7 o 17
Figu e 2. P ophage induc ion assays. (a) and (b)—PCR de ec ion o CJIE1 (a) and CCIE1 (b) ci cula
phagic DNA a ge ing CJIE1-CCIE1 phage mo phogenesis p o ein gene (513 bp amplicon) and he
bac e ial glu amine syn he ase gene (615 bp amplicon). Odd lanes e e o bac e ial gene ampli ica-
ion and e en lanes e e o phage gene ampli ica ion. The i s pai o each induc ion condi ion e e
o un ea ed DNA, while he second pai e e o Exonuclease I only and he hi d o Exonuclease I
and Lambda Exonuclease double- ea ed DNA. Resul s a e shown o non-supplemen ed con ol
(lanes 1–6), mi omycin C induc ion (lanes 7–12), and sodium deoxychola e induc ion (lanes 13–18).
M, 100 bp DNA Ladde (NEB). (a) PCR eac ions on DNA ex ac ed om concen a ed pu a i e
phage pa icles ob ained upon induc ion o he CJIE1-like p ophage. The ampli ica ion o he phagic
gene ollowing linea DNA elimina ion by exonuclease ea men sugges s he induc ion o his p o-
phage, e en in he non-supplemen ed con ol. Simila esul s we e ob ained o he induc ions o
he CJIE2-like p ophage (da a no shown). (b) PCR eac ions on DNA ex ac ed om concen a ed
pu a i e phage pa icles ob ained upon induc ion o he CCIE1-like p ophage. The lack o ampli i-
ca ion o he phagic gene ollowing linea DNA elimina ion by exonuclease ea men sugges s ha
no induc ion occu ed o his p ophage. Simila esul s we e ob ained o he induc ions o he
CCIE2-like p ophage (da a no shown). (c) and (d
)
—Nega i e s aining ansmission elec on mi-
c oscopy images ob ained a e induc ion o CJIE1 (c) and CJIE2 (d) p ophages. Roughly icosahed al
phage-like pa icles we e obse ed (a ows) wi h a diame e o 49 ± 4 nm (CJIE1, (c)) and 50 ± 3 nm
(CJIE2, (d)). Al hough se e al ail- ela ed p o eins we e anno a ed in he genome, no ail-like s uc-
u es we e obse ed on CJIE1 pa icles (c), and ail- esembling s uc u es we e inconsis en ly ob-
se ed o CJIE2 (whi e a ow head on (d)).
(a)
(b)
(c) (d)
Mic oo ganisms 2022, 10, 516 8 o 17
Nega i e s aining ansmission elec on mic oscopy was pe o med on PCR-posi i e
phage p ecipi a es, and phage-like s uc u es we e obse ed (a ows on Figu e 2C,D). Al -
hough sca ce, such s uc u es we e obse ed in all he PCR-posi i e phage p ecipi a es,
showing oughly icosahed al heads wi h 49 ± 4 nm (CJIE1—Figu e 2C) and 50 ± 3 nm
(CJIE2—Figu e 2D). E en hough se e al ail- ela ed p o eins we e anno a ed in he ge-
nome and p edic ed by VIRFAM, no ail-like s uc u es we e obse ed on CJIE1 pa icles
(Figu e 2C) and ail- esembling s uc u es we e inconsis en ly obse ed o CJIE2 (Figu e
2D, whi e a ow head). This ea u e may be due o he lack o ail- ela ed p o eins ende -
ing ailless phages o possibly due o some limi a ions on nega i e s aining which may
no e eal phage ails because o hei limi ed densi y [28]. The obse ed bac e iophage-
like s uc u es conjuga ed wi h he PCR de ec ion o ci cula phage DNA on cul u e su-
pe na an s upon induc ion poin s o he p oduc ion o CJIE1 and CJIE2 bac e iophage
pa icles, hus no suppo ing he p e ious epo s o CJIE1 and CJIE2 as incomple e, a
leas in he assayed Campylobac e spp. s ains.
3.4. Iden i ica ion o CJIE1-, CJIE4-, and CCIE2-Like P ophages wi hin Campylobac e spp.
To unde s and he phage dynamics on Campylobac e spp. popula ion, he e e ence
CJIE1-1 and CJIE4-1 p ophages, as well as he newly iden i ied CCIE2 p ophage, we e
used as empla e model p ophages, since he p ophages iden i ied using PHASTER and
PHT ell essen ially in hese h ee g oups (Figu e 3A). In eg a ed elemen s CJIE3 and
CJIE5 we e no included as models o his sc eening as nei he PHASTER- no PHT-p e-
dic ions e idenced any o hese p ophages. To double-check his, BLASTn que ying CJIE3
and CJIE5 we e conduc ed, iden i ying no CJIE3-like egions in he newly sequenced C.
coli and C. jejuni genomes, bu e i ying he p esence o CJIE5 in all genomes. Indeed,
CJIE5, which is likely no a p ophage, is also p esen in C. jejuni RM1221 (whe e all we e
ini ially iden i ied) and in C. jejuni NCTC11168 (desc ibed as a p ophageless s ain). The
o al numbe o p ophages ound was 177 (Tables 3 and S1). In de ail, he sc eening on he
104 genomes o his s udy ende ed a o al o 39 Campylobac e spp. isola es ca ying CJIE1-
like p ophages, 53 ca ying CCIE2-like p ophages, and 4 ca ying CJIE4-like p ophages.
Among hese genomes, 12.5% (13/104) ha bo ed wo p ophages, ei he CJIE1 and CJIE2
o CJIE2 and CJIE4 (consul Tables S1 and S4 o de ails). Sc eening on 736 publicly a ail-
able genomes ende ed a o al o 48 Campylobac e spp. isola es ca ying CJIE1-like p o-
phages, 24 ca ying CCIE2-like p ophages, and 9 ca ying CJIE4-like p ophages (Table 3).
Among hese genomes, 1.1% (8/736) ha bo ed wo p ophages, CJIE1 and CJIE2 (Table S5).
The p ophage p esence a io on Po uguese isola es was much highe han in he publicly
a ailable genomes o Campylobac e spp. (Table 3), and i does no appea o be ela ed
wi h ST o CC ype (Tables S1, S4 and S5). Di e en Campylobac e spp. popula ions, ei he
in e ms o loca ion, hos o isola ion, o pheno ypic cha ac e is ics, seem o di e en ially
ca y CJIE elemen s (Table S6), suppo ing a po en ial ole o hese p ophages in he mod-
ula ion o ca ie s genomic and pheno ypic ea u es [14,16,17,52], and in he e olu ion and
ecological adap a ion o he isola es (as e iewed by Ha ison and B ockhu s (2017) [53]).
Howe e , ou app oach o conside ing only p ophages wi h empla e co e age o e 50%
may ha e led o he disca d o p ophages, namely emnan , unca ed, o mosaic p o-
phages, which may ha e been analyzed in he p e iously men ioned s udies. None heless,
he e e se easoning is also alid: i is possible ha we ha e conside ed dis inc phages
o be simila o he e e ence phages due o sequence co e age abo e he 50% ma k. Fo
da abases- e ie ed isola es, we should emind ha only isola es wi h in o ma ion ega d-
ing MLST, coun y o isola ion, and hos o ganism we e selec ed, which may ha e led o
he non-analysis o p ophages e en ually ca ied by he emaining genomes a ailable in
he da abases.
Mic oo ganisms 2022, 10, 516 9 o 17
Figu e 3. Phylogene ic ee o he iden i ied CJIE1-like, CCIE2-like, and CJIE4-like p ophages (a)
and po en ial p ophages hos -jumps (b) and (c). CCIE2-like p ophages a e ep esen ed in g een,
CJIE1-like p ophages a e ep esen ed in ed, and CJIE4-like p ophages a e ep esen ed in blue. The
Mic oo ganisms 2022, 10, 516 16 o 17
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