Packaging o Dino oseobac e shibae DNA in o
Gene T ans e Agen Pa icles Is No Random
Ju¨ genTomasch
1,3,
*, Hui Wang
1
, Ap il T.K. Hall
2
,DianaPa zel
1,6
, Ma hias P eusse
3
,Jo¨ nPe e sen
4
,
Henne B inkmann
4
, Boyke Bunk
4
, Sabin Bhuju
5
,MichaelJa ek
5
, Robe Ge e s
5
, And ew S. Lang
2
,and
I ene Wagne -Do¨ble
1
1
G oup Mic obial Communica ion, Helmhol z-Cen e o In ec ion Resea ch, B aunschweig, Ge many
2
Depa men o Biology, Memo ial Uni e si y o New oundland, S John’s, New oundland and Lab ado , Canada
3
Depa men o Molecula Bac e iology, Helmhol z-Cen e o In ec ion Resea ch, B aunschweig, Ge many
4
Depa men o Mic obial Ecology and Di e si y Resea ch, Leibniz Ins i u e DSMZ—Ge man Collec ion o Mic oo ganisms and Cell Cul u es,
B aunschweig, Ge many
5
G oup Genome Analy ics, Helmhol z-Cen e o In ec ion Resea ch, B aunschweig, Ge many
6
P esen add ess: Sa o ius S edim Bio ech GmbH, Go¨ ingen,Ge many
*Co esponding au ho : E-mail: jue gen. omasch@helmhol z-hzi.de.
Accep ed: Janua y 8, 2018
Abs ac
Gene ans e agen s (GTAs) a e phage-like pa icles which con ain a agmen o genomic DNA o he bac e ial o
a chaeal p oduce and deli e his o a ecipien cell. GTA gene clus e s a e p esen in he genomes o almos all ma ine
Rhodobac e aceae (Roseobac e s) and migh be impo an con ibu o s o ho izon al gene ans e in he wo ld’s oceans. Fo
all o ganisms s udied so a , no ob ious e idence o sequence speci ici y o o he non andom p ocess esponsible o packaging
genomic DNA in o GTAs has been ound. He e, we show ha knock-ou o an au oinduce syn hase gene o Dino oseobac e
shibae esul ed in o e p oduc ion and elease o unc ional GTA pa icles (DsGTA). Nex -gene a ion sequencing o he 4.2-kb
DNA agmen s isola ed om DsGTAs e ealed ha packaging was no andom. DNA om low-GC conjuga i e plasmids bu no
om high-GC ch omids was excluded om packaging. Se en ch omosomal egions we e s ongly o e ep esen ed in DNA
isola ed om DsGTA. These packaging peaks lacked iden i iable conse ed sequence mo i s ha migh ep esen ecogni ion
si es o he GTA e minase complex. Low-GC egions o he ch omosome, including he o igin and e minus o eplica ion, we e
unde ep esen ed in DNA isola ed om DsGTAs. DNA me hyla ion educed packaging equency while he le el o gene
exp ession had no in luence. Ch omosomal egions ound o be o e - and unde ep esen ed in DsGTA-DNA we e egula ly
spaced. We p opose ha a “head ul” ype o packaging is ini ia ed a he si es o co e age peaks and, a e linea iza ion o he
ch omosomal DNA, p oceeds in bo h di ec ions om he ini ia ion si e. GC-con en , DNA-modi ica ions, and ch oma in s uc u e
migh in luence a which sides GTA packaging can be ini ia ed.
Key wo ds: gene ans e agen , GTA, Roseobac e , ho izon al gene ans e .
In oduc ion
P oka yo es, e en di e en s ains o one species, o en di e
ema kably in hei genomic in en o ies. This a iabili y in ge-
nome con en canno be explained by gene loss alone (Dagan
and Ma in 2007) bu is he esul o a conside able amoun o
ho izon al gene ans e (HGT), which has been shown o be a
main d i e o p oka yo e e olu ion (McIne ney e al. 2017).
Mechanisms o HGT in p oka yo es include ans o ma ion,
ha is, di ec up ake o naked DNA om he en i onmen ,
conjuga ional ans e o plasmids ha equi es di ec con ac
o dono and ecipien cells (Thomas and Nielsen 2005), and
ansduc ion by phages (Canchaya e al. 2003). In 1974 an
addi ional, unique way o HGT o genomic DNA, independen
o cell–cell con ac o he p esence o naked DNA in he en i-
onmen , was disco e ed in Rhodobac e capsula us (Ma s
1974). La e , he esponsible R. capsula us gene ans e agen
(RcGTA) was ound o esemble a small ailed phage ha con-
ains agmen s o cellula genomic DNA ins ead o i al DNA
ßThe Au ho (s) 2018. Published by Ox o d Uni e si y P ess on behal o he Socie y o Molecula Biology and E olu ion.
ThisisanOpenAccessa icledis ibu edunde he e mso heC ea i eCommonsA ibu ionNon-Comme cialLicense(h p://c ea i ecommons.o g/licenses/by-nc/4.0/),whichpe mi snon-
comme cial e-use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Fo comme cial e-use, please con ac jou nals.pe [email p o ec ed]
Genome Biol. E ol. 10(1):359–369. doi:10.1093/gbe/e y005 Ad ance Access publica ion Janua y 9, 2018 359
GBE
Downloaded om h ps://academic.oup.com/gbe/a icle-abs ac /10/1/359/4794729
by Ges Bio echnologische use
on 07 Feb ua y 2018
(Yen e al. 1979). Since hen, Gene ans e agen (GTA) pa -
icles o di e en e olu iona y o igins ha e been iden i ied in
a ious o he bac e ia (Humph ey e al. 1997;Bie s e al. 2008)
and he a chaeon Me hanococcus ol ae (Be ani 1999).
In Alphap o eobac e ia, wo phylogene ically un ela ed
ypes o GTAs ha e been iden i ied, namely he
Rhodobac e ales GTA, wi h he bes s udied RcGTA, and
he Ba onella GTA (BaGTA). Those ela ed o RcGTA ha e
been iden i ied in many Alphap o eobac e ia and a e pa ic-
ula ly widesp ead in membe s o he o de Rhodobac e ales
whe e hey a e o ganized in a conse ed gene clus e o ap-
p oxima ely 15 kb (Lang and Bea y 2007;New on e al.
2010;Huang e al. 2011). As RcGTA packages only app ox-
ima ely 4 kb o DNA i has o be conside ed non-sel - ans-
missible (Lang e al. 2017). GTAs isola ed om
Rhodobac e aceae o he Roseobac e g oup ans e ed e-
sis ance ma ke s wi h high equencies o bo h cul u es and
na u al communi ies o ma ine bac e ia, sugges ing an impo -
an ole o GTAs o he gene low in he wo ld’s oceans
(McDaniel e al. 2010). Fu he mo e, phylogenomic analysis
iden i ied GTAs as he main sou ce o genes acqui ed h ough
HGT in membe s o he genus Phaeobac e , consis ing mainly
o ma ine su ace colonize s (F eese e al. 2017).
The Rhodobac e ales GTAs p obably e ol ed om a p o-
phage o he Sipho i idae amily ha los genes o in eg a ion
in o and excision om he hos DNA as well as o eplica ion,
whe eas genes o DNA packaging and s uc u al componen s
we e e ained (Huang e al. 2011), and hese genes we e sub-
sequen ly b ough unde he con ol o he hos gene egula-
o y sys ems (Me ce e al. 2012).InR. capsula us,genes coding
o he compe ence sys em, needed o up ake o DNA om
GTAs by he ecei ing cell, a e unde con ol o he same eg-
ula o s as he GTA genes (B imacombe e al. 2015). Recen ly i
has been ound ha he RcGTA is induced by nu ien deple-
ion, possibly as a consequence o inc eased le els o
guanosine- e aphospha e (ppGpp) (Wes bye e al. 2017).
No ably, only a small subse o a R. capsula us cul u e (<3%)
exp esses RcGTA genes, leading o lysis o he p oducing cells
(Fogg e al. 2012;Hynes e al. 2012).
A second phylogene ically dis inc alphap o eobac e ial
GTA has been ound in he in acellula bac e ia om he
genus Ba onella (Be glund e al. 2009;Tama i e al.2017).
As in R. capsula us, BaGTA p oduc ion is limi ed o a small
ac ion o he popula ion and hese p oduce s lyse o elease
GTA pa icles (Queba e e al. 2017). The au ho s sugges ed
ha he i es subpopula ion o B. henselae cells, indica ed
by low le els o ppGpp, sac i ices i sel o sp ead bene icial
mu a ions ia GTAs h oughou he popula ion o ac i ely
di iding cells.
The mechanism o packaging DNA in o GTA pa icles is
cu en ly no known. Based upon homology o key compo-
nen s like e minase and po al p o eins i is assumed ha i
esembles he “head ul” ype o packaging ha is used by
many double-s anded DNA phages (Lang e al. 2017). The
pa s o he genomes ha a e mobilized by packaging in o
GTAs ha e been de e mined o R. capsula us as well as B.
g ahamii and B. henselae. These s udies sugges andom
packaging o hos genomic DNA in o GTAs.
Ma ke ans e and DNA diges ion analysis in he p ege-
nomic e a sugges ed andom packaging o genomic DNA in o
RcGTAs (Ma s 1974;Humph ey e al. 1997;Be ani 1999).
These ini ial indings la e gained suppo by a s udy employ-
ing whole-genome mic oa ay hyb idiza ion o RcGTA-DNA,
which showed ha only he GTA gene clus e i sel was un-
de ep esen ed, by 25%, in packaged DNA, whe eas he
emaining genomic DNA was andomly packaged (Hynes
e al. 2012). Since he GTA clus e is highly ansc ibed in
he subse o he popula ion ha is esponsible o p oducing
he GTA pa icles, i was hypo hesized ha access o he GTA
encoding DNA was blocked by he RNA polyme ase complex
(Hynes e al. 2012).
Fo BaGTA, a i ulence egion impo an o hos -
adap abili y o he mouse pa hogen B. g ahamii was ound
o be o e ep esen ed in GTA-DNA (Be glund e al. 2009).
Genomic DNA o his egion was p esen in highe copy num-
be in he cells due o un-o eplica ion om a phage-de i ed
o igin o eplica ion, hus packaging in his case can be
conside ed andom bu dependen on he copy numbe o
he DNA (Be glund e al. 2009). A ecen ansposon se-
quencing s udy in B. henselae con i med he ela ionship
be ween DNA copy numbe and equency o packaging
o ch omosomal DNA bu addi ionally showed ha DNA
om plasmids is no ans e ed by BaGTAs (Queba e
e al. 2017).
The genome o he dino lagella e-associa ed bac e ium
Dino oseobac e shibae consis s o a 3.79-Mb ch omosome
and i e ex ach omosomal eplicons (Wagne -Do¨ ble e al.
2010). Two o hem (153 and 72 kb) ha e a simila high GC-
con en (65–68%) and codon-usage as he ch omosome and
a e he e o e classi ied as “ch omids”’ acco ding o Ha ison
e al. (2010). The o he h ee (191, 126, and 86 kb) ha e a
lowe GC-con en (60–61%) and de ia ing codon-usage and
ep esen ypical plasmids (Pe e sen e al. 2013). The 191- and
126-kb plasmids ha bo ype IV sec e ion sys ems ha a e
employed o hei conjuga ional ans e (Pa zel e al.
2016). A comple e GTA gene clus e is p esen on he D.
shibae ch omosome (Wang e al. 2014). Simila o R. capsu-
la us (Me ce e al. 2012), he D. shibae GTA (DsGTA) gene
clus e is unde he con ol o he C A phospho elay (Wang
e al. 2014). This signal- ansduc ion sys em is in eg a ed in o
he D. shibae quo um sensing (QS) sys em as i is induced by
he p oduc o he au oinduce syn hase LuxI
1
(Pa zel e al.
2013) and hen induces exp ession o wo addi ional QS e-
sponse egula o /au oinduce syn hase ope ons, luxR/I
2
,and
luxB/I
3
(Wang e al. 2014). He e, we show ha knockou o he
au oinduce syn hase gene luxI
2
esul s in o e p oduc ion o
DsGTAs. Using nex -gene a ion sequencing, we analyze pack-
aging o D. shibae DNA in o GTA pa icles.
Tomasch e al. GBE
360 Genome Biol. E ol. 10(1):359–369 doi:10.1093/gbe/e y005 Ad ance Access publica ion Janua y 9, 2018
Downloaded om h ps://academic.oup.com/gbe/a icle-abs ac /10/1/359/4794729
by Ges Bio echnologische use
on 07 Feb ua y 2018
Ma e ials and Me hods
Bac e ial S ains and Plasmids
Dino oseobac e shibae s ains used in his s udy, E. coli
s ains, plasmids and p ime s used o cons uc ion o he D.
shibae DluxI
2
(Dshi_2852) dele ion s ain a e lis ed in supple-
men a y able S1,Supplemen a y Ma e ial online. D. shibae
DluxI
2
was gene a ed by eplacing he gene wi h a gen amy-
cin esis ance casse e ia double homologous ecombina ion
as p e iously desc ibed (Pa zel e al. 2013). The knock-ou
ec o pJB5603luxI
2
:: Gm
was subcloned in E. coli ST18 and
in oduced in o D. shibae DFL12 ia conjuga ion.
Resequencing o he knock-ou s ain con i med dele ion o
he wild- ype gene, inse ion o he esis ance casse e in o
he co ec locus and iden i ied one seconda y mu a ion.
De ails on knock-ou cons uc ion and con i ma ion can be
ound in supplemen a y me hods, no e S1 and igu e S1,
Supplemen a y Ma e ial online.
Cul i a ion
Dino oseobac e shibae s ains we e g own in 1=2Di co
Ma ine B o h 2216 (BD, Spa ks, MD) o a i icial seawa e
medium (ASM, supplemen a y me hods)a 30Cand
160 pm. Ruege ia pome oyi was g own in 1=2yeas ex ac -
yp one-sea sal (YTSS) medium (4 g l
1
yp one, 2.5 g l
1
yeas ex ac , and 15 g l
1
sea sal s) a 20 C and 160 pm.
De ec ion o he GTA Majo Capsid P o ein by Wes e n
Blo
Dino oseobac e shibae s ains we e g own o 19 h in ASM.
Samples om Ruege ia pome oyi,g ownin1=2YTSS o 19 h,
we e included as posi i e con ols as his o ganism is known
o p oduce GTA pa icles (Bie s e al. 2008) and has de ec able
GTA capsid p o ein (Fu e al. 2010). Cells om 0.4 ml o each
cul u e we e pelle ed and esuspended in an equal olume o
TE bu e . Cul u e supe na an s we e p epa ed om cul u es
by cen i uga ion o 2 min a 17,000 g, wi h emo al o a
ac ion o he esul ing supe na an o a new ube. The sam-
ples (5 and 10 ll o cells and supe na an s, espec i ely) we e
mixed wi h SDS-PAGE sample bu e (NEB) and incuba ed
5mina 98C be o e elec opho esis. SDS-PAGE and immu-
noblo ing we e pe o med as desc ibed p e iously (Fu e al.
2010) using he p ima y an ibody a ge ing a egion o he
GTA majo capsid p o ein (Ag ise a AB, V€
ann€
as, Sweden) ha
is conse ed among many Rhodobac e ales.Theimageswe e
cap u ed using he ImageQuan LAS 4000 ins umen (GE Li e
Sciences, Uppsala, Sweden) and we e each uni o mly ad-
jus ed o b igh ness and con as .
Gene T ans e Bioassays
Gene ans e agen dono s ains we e g own o 24 h in
ASM a 30 C and 160 pm. The ecipien s ain, D. shibae
DFL12, was g own unde he same condi ions in 1/2 Mb. The
dono cul u es we e passed h ough 0.22-mm il e sand
0.5 ml o his il a e was added o 0.1 ml o he ecipien cells.
The mix u e was hen incuba ed a 30 C o 1 h wi hou
shaking, o 4 h and 160 pm, ollowed by addi ion o
0.9 ml ASM and a u he incuba ion a 30 C and 160 pm
o 18 h. The cells we e hen pla ed on 1/2 Mb wi h gen a-
mycin sul a e (150 mgml
1
) and incuba ed a 30 C o
2–4 days un il colonies we e isible o coun ing. The nega i e
con ols o hese assays we e il a es o he gen amycin-
sensi i e s ain, DFL12, and he gen amycin- esis an s ain,
DluxI
1
, added o he ecipien cells. Fil a e-only con ols ha
con ained no ecipien cells we e also included o con i m no
cells passed h ough he 0.22-mm il e s.
Pu i ica ion o DsGTA Pa icles o Elec on Mic oscopy
Dino oseobac e shibae DluxI
2
was g own 1/2 Mb o 29 h a
30 C wi h shaking a 160 RPM. The cells we e pelle ed by
cen i uga ion and NaCl was added o he supe na an o a
inal concen a ion o 1 M. Polye hylene glycol (PEG) 6000 was
added o make a inal concen a ion o 10% w/ and dissol ed
by s i ing a oom empe a u e. The solu ion was hen incu-
ba ed a 4 C o 18 h and he p ecipi a ed ma e ial pelle ed by
cen i uga ion a 10,000 g o 20mina 20C. The supe -
na an was pou ed o and pelle ed ma e ial was esuspended
in 0.1 M ammonium ace a e (pH 7) wi h shaking a oom em-
pe a u e o 2 h ollowed by gen le pipe ing. This ma e ial was
cen i uged a 2,400 g o 2 min a oom empe a u e and
he supe na an collec ed in o a new ube. T i on X-100 was
added o a inal concen a ion o 10% ( / ) wi h gen le mixing
by in e sion. This was cen i uged a 1,10,000 g o 2 h a 20
C, and mos o he supe na an hen emo ed. The pelle was
esuspendedin he emainingsolu ion(0.5 ml) by gen le
pipe ing and he ube e illed wi h 0.1 M ammonium ace a e.
The ube was cen i uged again o 1 h and he p e ious s eps
epea ed. A e his hi d cen i uga ion, almos all o he su-
pe na an was emo ed and he pelle le o esuspend in he
small amoun o emaining liquid wi h shaking a 4 C o 18 h.
The ma e ial was homogenized by gen le pipe ing, ans-
e ed o a mic o uge ube, and cen i uged a 17,000 g
o 1 min a oom empe a u e. The supe na an was collec ed
o elec on mic oscopy imaging a he Uni e si y o Guelph
(Canada) Molecula and Cellula Imaging Facili y. A 5-mlsample
was placed on o a 200 mesh coppe g id wi h o m a /ca bon
coa ing, adso bed o 1 min, and blo ed o wi h il e pape .
The g id was hen loa ed on a d op o 2% u anyl ace a e. The
sample was iewed in a FEI Tecnai F20 elec on mic oscope a
200kV.Theimageswe ecollec edwi haGa an4Kbo om-
moun came a using he Ga an Digi alMic og aph so wa e.
Isola ion o DsGTA-DNA
DsGTA-DNA was isola ed om h ee independen cul u es.
The cul u es we e ea ed as o he Elec on mic oscopy up o
Gene T ans e Agen s o Dino oseobac e shibae GBE
Genome Biol. E ol. 10(1):359–369 doi:10.1093/gbe/e y005 Ad ance Access publica ion Janua y 9, 2018 361
Downloaded om h ps://academic.oup.com/gbe/a icle-abs ac /10/1/359/4794729
by Ges Bio echnologische use
on 07 Feb ua y 2018
he poin o esuspension o he PEG p ecipi a es. In his case,
he pelle s we e esuspended in TE bu e wi h gen le shaking
a oom empe a u e o 2 h. F ee nucleic acids we e emo ed
by ea men wi h DNase I (0.01 U ll
1
) and RNase A
(0.08 U ll
1
)a 37Cin1DNase bu e (New England
Biolabs) o 1 h. The sample was hea ed a 75 C o 15min
a e addi ion o EDTA o a inal concen a ion o 5 mM.
Nucleic acids we e pu i ied by phenol: chlo o o m: isoamyl
alcohol (25:24:1) ex ac ion and e hanol p ecipi a ion. The
pu i ied DNA was subjec ed o es ic ion enzyme diges ion
using he enzymes EcoRI and B aI (New England Biolabs),
acco ding o he manu ac u e ’s p o ocol. The un ea ed
DNA sample was incuba ed unde he same condi ions (i.e.,
in 1 eac ion bu e a 37 C) wi h he addi ion o dH
2
Oin
place o enzyme. The app oxima ely 4.2-kb band o DsGTA-
DNA was excised om he gel and pu i ied wi h he Wiza d
SV Gel and PCR Clean-Up Sys em (P omega, Fi chbu g, WI),
ollowed by e hanol p ecipi a ion.
Isola ion o Genomic DNA
Genomic DNA was isola ed om 5 ml samples o wo o he
D. shibae cul u es used o isola ion o DsGTA-DNA using he
NucleoSpin issue ki (Mache y-Nagel, Du¨ en, Ge many)
acco ding o manu ac u e ’s p o ocols.
Isola ion o RNA
RNA o ansc ip ome analysis o he h ee di e en D. shibae
s ains was isola ed om wo independen cul u es. RNA o
analysis o he ela ionship o DsGTA packaging and ansc ip-
ion was isola ed om wo cul u es om which DsGTA-DNA
was isola ed. Cells we e collec ed by cen i uga ion a 12,000
g o 1mina 4C, co e ed wi h 1 ml T izol (Ambion,
Ge many), immedia ely ozen in liquid N
2
and s o ed a
70 C un il p ocessing. Fo RNA ex ac ion, cells we e ho-
mogenized wi h 0.3 g o glass beads in he Fas P ep-24 in-
s umen (MP Biomedicals, CA) a 6.0 m/s o 3 min and hen
incuba ed o 5 min a oom empe a u e. Samples we e cen-
i uged a 12,000 g o 10 min a 4 C and he supe na-
an s we e ans e ed o esh ubes, ollowed by addi ion o
100 ml o 1-b omo-3-chlo op opane (Sigma-Ald ich, S . Louis,
MO) and incuba ion o 10 min a oom empe a u e.
Samples we e cen i uged a 12,000 g o 10 min a 4 C,
a e which he aqueous phase was ans e ed o new ubes
and mixed wi h 500 ml o absolu e e hanol. Ex ac s we e pu-
i ied using RNeasy Mini ki (Qiagen, Hilden, Ge many)
acco ding o he manu ac u e ’s ins uc ions. In addi ion,
samples we e ea ed wi h DNAse I (Qiagen, Hilden,
Ge many).
Sequencing o RNA
To al RNA was ea ed wi h RiboZe o (Bac e ia) ki (Illumina,
San Diego, CA) ollowing he manu ac u e ’s p o ocol in
o de o emo e ibosomal RNA om he samples. Single
end, s and speci ic cDNA lib a ies we e p epa ed om
RNA deple ed o al RNA using Sc ip seq 2 RNA-Seq
Lib a y P epa a ion Ki (Illumina) ollowing he manu ac u e s
p o ocol. Fo sequencing equal olume o lib a ies (12 PM)
was mul iplexed on a single lane. Sequencing was done on
he HiSeq 2500 (Illumina) using T uSeq SBS Ki 3—HS
(Illumina) o 50 cycles esul ing in 50-bp eads. Image analysis
and base calling we e pe o med using he Illumina pipeline
1.8 (Illumina).
Sequencing o Genomic and DsGTA-DNA
Pai ed end cDNA lib a ies we e p epa ed using he NEBNex
Ul a DNA Lib a y P epa a ion Ki o Illumina (NEB, Ipswich,
MA). Sequencing was done on he MiSeq 2000 (Illumina)
using MiSeq Reagen Ki s 2 (Illumina) o 250 cycles esul ing
in 2250 bp pai ed end eads. Image analysis and base calling
we e pe o med using he Illumina pipeline 1.8 (Illumina).
PacBio-Sequencing and DNA Modi ica ion Calling
PacBio-sequencing was pe o med as p e iously desc ibed
(Ba ling e al. 2017). Me hylome analysis was pe o med using
he “RS_Modi ica ion_and_Mo i _Analysis.1” p o ocol in-
cluded in SMRT Po al e sion 2.3.0. 99.5 and 96.6% o he
iden i ied m6A and m4C mo i s we e me hyla ed, espec i ely.
P ocessing o Sequence Da a
The demul iplexed aw as q- iles we e quali y-con olled us-
ing he FASTQ-mc sui e (h ps://gi hub.com/Exp ession
Analysis/ea-u ils; las accessed Janua y 15, 2018). Low quali y
bases (Ph ed-sco e <30) and iden i ied Illumina adap o s
we e clipped om he sequences. Reads we e mapped o
e e ence genomes using bow ie2 (Langmead and Salzbe g
2012) wi h de aul pa ame e s o single o pai ed end eads.
Ambiguously mapping eads we e andomly dis ibu ed be-
ween all egions o which hey could be assigned. The esul -
ing sam- iles we e con e ed o indexed bina y o ma and
pile-up o ma using sam ools (Li e al. 2009). Accession num-
be s o e e ence D. shibae DNA sequences: ch omosome,
3.79 Mb [NC_009952.1]; pDSHI01, 191 kb [NC_009955.1];
pDSHI02, 153 kb [NC_009956.1]; pDSHI03, 126 kb
[NC_009957.1]; pDSHI04, 86 kb [NC_009958.1]; pDSHI05,
72 kb [NC_009959.1].
S a is ical Analysis
Pile-up iles we e loaded in o he R s a is ical en i onmen (R
e sion 3.4.0). Rep oducibili y o biological eplica es was
assessed by isual inspec ion o sca e plo s and boxplo s
and by calcula ing Spea man ank co ela ion (supplemen a y
igs. S7–S9,Supplemen a y Ma e ial online). Dis ibu ion o
DsGTA and genomic co e age was compa ed wi h heo e ical
da a ollowing a no mal dis ibu ion using he qqno m()
Tomasch e al. GBE
362 Genome Biol. E ol. 10(1):359–369 doi:10.1093/gbe/e y005 Ad ance Access publica ion Janua y 9, 2018
Downloaded om h ps://academic.oup.com/gbe/a icle-abs ac /10/1/359/4794729
by Ges Bio echnologische use
on 07 Feb ua y 2018
unc ion in R. The mean co e age by DsGTA-DNA, genomic
DNA, and RNA as well as he mean GC con en we e calcu-
la ed o sliding windows o 4 kb using he ollapply unc ion
o he zoo package (Zeileis and G o hendieck 2005). GC-
con en o sliding windows was calcula ed using he
le e F equency unc ion o he Bios ings package (Page`s e
al. 2017). Only me hyla ed si es wi h an iden i ica ion Ph ed
sco e >30 (99.9% p obabili y o co ec iden i ica ion) we e
used in his s udy and summed up o he sliding window
ange. Spea man ank co ela ion was calcula ed o DsGTA
co e age wi h GC-con en and log
2
- ans o med RNA co e -
age, espec i ely. The au oco ela ion unc ion, a ailable om
he R s a s package, calcula es he Pea son co ela ion om all
pai s o obse a ions ha ha e he same dis ance om each
o he . This is done o inc easing dis ances and makes i pos-
sible o ind dis ances a which he co ela ion o wo obse -
a ions (in his case DsGTA co e age) peaks, indica ing
pe iodici y o he da a. Cumula i e GC/AT-skew o iden i i-
ca ion o he o igin and e minus o eplica ion has been
pe o med using he o iloc unc ion o he seqin package
(Cha i and Lob y 2007).
Code A ailabili y
Linux shell and R sc ip s o analysis a e a ailable a h ps://
gi hub.com/Jue gen 79/GTA; las accessed Janua y 15, 2018.
Iden i ica ion o Phage Genes
The ool PHAST (Zhou e al. 2011) was used o iden i y phage
de i ed genes in he genome o D. shibae.
DNA Mo i Disco e y
DNA sequence o he se en DsGTA co e age peaks was used
as p ima y inpu o he mo i disco e y algo i hm o he
MEME sui e 4.12.0 (Bailey e al. 2009). Mo i disco e y was
un in he disc imina i e mode, wi h he se en peak egions
indica ed in igu e 3Aas que y inpu and ei he he whole
ch omosomal DNA sequence o DNA sequences o ch omo-
somal egions wi h co e age be ween 200 and 500 eads/n
as con ols (seconda y) inpu . The second con ol a oided in-
clusion o low GC egions ha showed an ex emely low
co e age.
Da a A ailabili y
RNA, genomic, and DsGTA Illumina sequencing da a ha e
been made publicly a ailable a he Eu opean Nucleo ide
A chi e (ENA, h p://www.ebi.ac.uk/ena; las accessed
Janua y 15, 2018) unde he p ojec accession numbe
PRJEB20656. PacBio da a used o e alua ion o D. shibae
DluxI
2
(supplemen a y ig. S1 and no e S1, Supplemen a y
Ma e ial online) has no been deposi ed in a public da abase
ye bu will be a ailable om he au ho s upon eques .
Resul s
Only weak exp ession o he DsGTA gene clus e could be
obse edinwild ypeD. shibae and i ually no exp ession
was ound in he luxI
1
knockou . Howe e , when we knocked
ou he C A-con olled luxI
2
gene (supplemen a y ig. S1 and
no e S1, Supplemen a y Ma e ial online), s ong exp ession o
he DsGTA gene clus e was obse ed ( ig. 1A). The DsGTA
majo capsid p o ein could acco dingly be de ec ed in cell
ex ac s and supe na an s o D. shibae DluxI
2
, indica ing
ha his s ain p oduces and eleases GTA pa icles. A weak
signal was also obse ed in cell-ex ac s, bu no supe na an s
o he wild ype, indica ing a low GTA p oduc ion a e in he
na i e s ain unde he condi ions s udied ( ig. 1B). The gen-
amicin esis ance casse e used as he ma ke o knock-ou
cons uc ion could be success ully ans e ed by cell- ee
supe na an s o D. shibae DluxI
2
, bu no DluxI
1
, o he wild
ype demons a ing ha DsGTAs a e unc ional and ha D.
shibae cells a e able o ecei e and inco po a e DNA om he
GTAs ( ig. 1C). A gen amycin- esis an s ain ob ained ia
GTA-media ed ma ke ans e also p oduced and eleased
inc eased numbe s o GTAs ela i e o he wild ype s ain,
alida ing he ole o he luxI
2
gene in ep essing DsGTA p o-
duc ion ( ig. 1D). DsGTA esembles a small ailed phage wi h
a head diame e o app oxima ely 33 nm and a ail leng h o
app oxima ely 48 nm ( ig. 1E). I packages DNA o 4.2 kb in
size, simila o RcGTA-DNA. T ea men wi h es ic ion
enzymes p oduced smea ing below his band, indica ing
ha he DNA was double-s anded and ha i was no a
speci ic sequence o DNA ( ig. 1F).
We used nex -gene a ion sequencing o analyze whe he
packaging o DNA in o DsGTA pa icles is andom, o shows
p e e ences o dis inc genomic loci. To his end, DsGTA-
DNA, genomic DNA, and RNA om he same samples we e
sequenced and mapped on he D. shibae genome. This
allowed us o de e mine he ela i e copy numbe o epli-
cons, o add ess he possibili y o un-o eplica ion ound in
in B. g ahamii and B. henselae (Be glund e al. 2009;Queba e
e al. 2017), and o s udy he ela ionship be ween ansc ip-
ional ac i i y and packaging equency simila o R. capsula us
(Hynes e al. 2012). Fu he mo e, we used PacBio sequencing
da a o de e mine me hyla ion o he D. shibae DNA. We
calcula ed he mean co e age o sliding windows o 4 kb,
o he ch omosome and ex ach omosomal eplicons.
Co e age o he mul ipa i e genome o D. shibae by DNA
isola ed om DsGTA pa icles showed a clea de ia ion om
no mal dis ibu ion and a long ail o lowe co e age
( ig. 2A). Co e age by genome sequencing o he acco ding
s ains mos ly ollowed a no mal dis ibu ion, wi h only ew
egions being o e o unde ep esen ed (supplemen a y ig.
S2,Supplemen a y Ma e ial online).
Co e age dis ibu ion di e ed o he six D. shibae epli-
cons ( ig. 2Band supplemen a y able S1,Supplemen a y
Ma e ial online). The ch omosome showed he highes
Gene T ans e Agen s o Dino oseobac e shibae GBE
Genome Biol. E ol. 10(1):359–369 doi:10.1093/gbe/e y005 Ad ance Access publica ion Janua y 9, 2018 363
Downloaded om h ps://academic.oup.com/gbe/a icle-abs ac /10/1/359/4794729
by Ges Bio echnologische use
on 07 Feb ua y 2018

a iabili y (be ween 10 and 763 eads/n , median: 181 eads/
n ); he 72-kb ch omid had a co e age simila o he ch omo-
some (median: 192 eads/n ) while he 153-kb ch omid
showed he highes abundance wi hin DsGTA-DNA (median:
1082 eads/n ). Co e age o he plasmids was low o e all
(median be ween 4 and 6 eads/n ), bu indica ed ha a leas
pa s o he 191- and 86-kb plasmids we e p esen in DsGTA-
DNA (maximum: 237 and 258 eads/n , espec i ely) whe eas
he 126-kb plasmid was almos absen (maximum: 8 eads/
n ). In he case o he 153-kb ch omid, he high co e age
could pa ly be explained by he 50% highe copy numbe
o his eplicon, whe eas he copy numbe o all o he epli-
cons was close o one ch omosome equi alen ( ig. 2C). Nex ,
we analyzed he co e age o each eplicon in de ail, s a ing
wi h he ex ach omosomal eplicons. Di e ences in DNA
con en o a sequencing bias could be excluded as a sou ce
explaining co e age di e ences o ch omids and plasmids
( ig. 2D). Bo h ch omids we e comple ely, albei no e enly,
co e ed by GTA-DNA. In pa icula , co e age o he 153-kb
ch omid showed a conspicuous gap be ween 95 and 102 kb.
The 126-kb plasmid showed i ually no mapping eads and
hus can be conside ed no o be packaged in o DsGTA pa -
icles. The 191 and 86 kb showed a simila low co e age ex-
cep o one 7.5-kb egion wi h e y high co e age and sha p
bounda ies. We ound ha he DNA sequences o hese
egions we e iden ical o he low co e age egion on he
153-kb ch omid. I con ains a ansposable elemen wi h
he gene hiC in he cen e ha has been sp ead o he di -
e en eplicons ( hiC locus, supplemen a y ig. S3A,
Supplemen a y Ma e ial online). Thus, andom mapping
should lead o an app oxima ely equal dis ibu ion o he am-
biguous eads be ween he h ee pa alogous a ge loci.
Assigning all he eads o he locus on he 153-kb ch omid
would ill he gap on his ch omid and esol e obse ed dis-
c epancies o ead mapping (supplemen a y ig. S3 and no e
S2, Supplemen a y Ma e ial online). The e o e, we conclude
ha he la ge majo i y o eads assigned o he hiC locus
o igina ed om he 153-kb ch omid, and ha plasmids a e
i ually excluded om being packaged in o GTA pa icles.
Plasmids showed a 60% and 40% highe equency o
FIG.1.—Biosyn hesis o unc ional GTAs in D. shibae DluxI
2
.(A) Exp ession o he DsGTA gene clus e in D. shibae DFL12, DluxI
1
,andDluxI
2
as
de e mined by s and-speci ic RNA-seq. Co e age o he nega i e s and is shown. DsGTA genes conse ed in Rhodobac e ales a e displayed as g ey a ows.
Genes a e numbe ed acco ding o he e e ence R. capsula us; p edic ed unc ions a e indica ed on he igh . Rep oducibili y o RNA sequencing is shown in
supplemen a y ig. S7,Supplemen a y Ma e ial online. (B) De ec ion o he GTA majo capsid p o ein in cell ex ac s and cul u e supe na an s o Ruege ia
pome oyi (Rp, posi i e con ol) and he a o emen ioned D. shibae s ains using wes e n blo ing. (C) T ans e o a gen amycin- esis ance casse e o D. shibae
DFL12 by supe na an s om di e en D. shibae s ains. Indi idual da a om h ee (DFL12, DluxI
2
)and wo(DluxI
1
) independen expe imen s is shown. (D)
T ans e o he gen amycin- esis ance casse e dis up ion o luxI
2
ia GTA esul s in o e p oduc ion o GTAs in he esul ing ecipien s ain (AH1). (E) Elec on
mic og aph showing wo DsGTA pa icles. The ilamen ous s uc u es a e mos likely lagella. (F) Visualiza ion o he DNA isola ed om GTAs: un ea ed DNA
(U) and DNA diges ed wi h BamHI (B) and EcoRI (E) a e shown.
Tomasch e al. GBE
364 Genome Biol. E ol. 10(1):359–369 doi:10.1093/gbe/e y005 Ad ance Access publica ion Janua y 9, 2018
Downloaded om h ps://academic.oup.com/gbe/a icle-abs ac /10/1/359/4794729
by Ges Bio echnologische use
on 07 Feb ua y 2018
me hyla ed adenosines wi hin 4-kb sliding windows han he
ch omids and he ch omosome, espec i ely. The equency
o me hyla ed cy osines was simila o all eplicons (supple-
men a y ig. S4 and ables S2 and S3, Supplemen a y Ma e ial
online).
The ch omosome showed a clea ly non andom co e age
wi hin he DsGTA-DNA ( ig. 3A). In con as , genome se-
quencing demons a ed a egula pa e n o co e age and
hus un-o eplica ion, o some o he cause o di e ences
in he amoun s o di e en egions o he ch omosome being
p esen in cells, could be excluded as a po en ial explana ion
o he obse ed pa e n in he DsGTA-DNA co e age
( ig. 3A). Fou almos egula ly spaced egions we e clea ly
unde ep esen ed, including he o igin and e minus o epli-
ca ion ( ig. 3A,supplemen a y ig. S5,Supplemen a y
Ma e ial online). In addi ion, he GTA gene clus e i sel
showed e y low packaging. In con as , se en egions we e
p e e en ially packaged in o DsGTA pa icles. Be ween hose
clea ly o e - and unde ep esen ed egions, se e al local max-
ima and minima exis . Regions wi h low co e age ended o
ha e a low GC-con en ( ig. 3B), consis en wi h he low co -
e age o he low-GC plasmids. The cooccu ence o local min-
ima in GC-con en and GTA packaging was mos ob ious o
he o igin and e minus o eplica ion. A local minimum in
GTA co e age wi hin peak 4 could be obse ed coinciding
wi h a local minimum o GC-con en ( ig. 3A). On he o he
hand, less equen packaging o he GTA clus e could no be
explained by GC-con en . O e all, he s ong posi i e co ela-
ion be ween packaging and GC-con en was highly signi i-
can ( ig. 3D). We could no iden i y DNA mo i s such as
possible ecogni ion si es o es ic ion enzymes, phage-like
ecogni ion si es, o epea s ha we e en iched in egions
wi h high packaging a e using he MEME sui e o sequence
mo i disco e y. Besides he GTA gene clus e , one de ec i e
phage egion, lacking a phage eplicase gene, was iden i ied
in he D. shibae genome a posi ion 98.7–102.8 kb, jus ou -
side peak 1 (supplemen a y able S4,Supplemen a y Ma e ial
online). Genes encoding ansposable elemen s o RNAs
we e also no ound wi hin he DsGTA co e age peaks (sup-
plemen a y able S5,Supplemen a y Ma e ial online). The o -
igin and e minus o eplica ion showed a highe equency o
me hyla ed adenosines (m6A) ( ig. 3C), whe eas he e-
quency o me hyla ed cy osines (m4C) lacked such a pa e n
(supplemen a y ig. S6,Supplemen a y Ma e ial online). The
nega i e co ela ion be ween packaging in o DsGTAs and he
numbe o m6A-si es was o e all lowe han o he
FIG.2.—Packaging o DNA o igina ing om di e en eplicons. (A) Quan ile-quan ile plo compa ing log
10
- ans o med sequencing ead-co e age o
DsGTA-DNA o a heo e ical no mal dis ibu ion (uppe panel) and his og am showing dis ibu ion o log
10
-co e age (lowe panel). Mean alues o 4-kb bins
o e lapping by 2-kb a e shown. Red line in he uppe panel indica es no mal dis ibu ed da a. (B) Dis ibu ion o log
10
-co e age o DsGTA-DNA by eplicon.
A e age GC-con en o each eplicon is indica ed. (C) Dis ibu ion o log
10
-co e age o D. shibae genomic DNA by eplicon. The boxplo s p esen ed in B
and Cshow he 25 h and 75 h qua iles as whiske s, he in e qua ile ange as box and he median as line. These da a ha e been ep oduced (supplemen a y
ig. S8,Supplemen a y Ma e ial online). (D) Co e age o ex ach omosomal eplicons by sequencing eads om DsGTA-DNA (blue and ed, acco ding
o ype o eplicon) and genomic DNA (g ey). Posi ion o a pa alogous egion o which eads mapped ambiguously is indica ed by an as e isk (supplemen a y
ig. S3 and no e S2, Supplemen a y Ma e ial online).
Gene T ans e Agen s o Dino oseobac e shibae GBE
Genome Biol. E ol. 10(1):359–369 doi:10.1093/gbe/e y005 Ad ance Access publica ion Janua y 9, 2018 365
Downloaded om h ps://academic.oup.com/gbe/a icle-abs ac /10/1/359/4794729
by Ges Bio echnologische use
on 07 Feb ua y 2018
co ela ion wi h he GC-con en ( ig. 3E). Gene exp ession did
no ha e a la ge in luence on packaging equency, nei he
iewed globally o bo h s ands ( ig. 3D) no when exp ession
o genes by s and was in es iga ed (supplemen a y ig. S6,
Supplemen a y Ma e ial online). Only a weak, bu s ill signi i-
can , nega i e co ela ion was ound ( ig. 3D). Wi hin peaks 3,
4, and 6, local minima co esponded o highly exp essed genes
( ig. 3Aand supplemen a y able S5,Supplemen a y Ma e ial
online). The i s imp ession o a egula spacing o he di e en
packaging equency egions gained suppo om an au oco -
ela ion analysis. Fo his me hod, adap ed om ime-se ies
analysis, Pea son co ela ion is calcula ed om all pai s o co -
e age alues ha ha e he same dis ance om each o he and
his is done o inc easing dis ances. The esul ing plo , showing
he co ela ion o de ed by inc easing dis ance, indica ed a sig-
ni ican pe iodici y o he DsGTA co e age da a wi h maximal
co ela ion a dis ances sligh ly less han one qua e , one hi d,
and hal he ch omosome size ( ig. 3F).
Discussion
He e, we demons a ed ha knockou o he au oinduce
syn hase-encoding gene luxI
2
leads o o e p oduc ion o
GTA pa icles by D. shibae. Thus, he p oduc o LuxI
2
likely
ac s as a ep esso o GTA gene exp ession. T ansc ip ion o
bo h he GTA gene clus e as well as he ope on coding o
LuxI
2
and he AHL-binding ansc ip ion ac o LuxR
2
is in-
duced by he same egula o y sys em, he C A phospho elay
(Wang e al. 2014). Coac i a ion o he GTA gene clus e and
i s ep esso migh o e an explana ion o bis abili y o GTA
gene exp ession wi h only a subse o cells p oducing GTA
pa icles (Fogg e al. 2012;Hynes e al. 2016;Queba e e al.
FIG.3.—Packaging o ch omosomal DNA in o GTAs in compa ison o ch omosomal posi ion, GC-con en , and ansc ip ion. (A) Co e age o he
ch omosome by DsGTA (black) and genomic DNA (g ey). O e and unde ep esen ed egions a e highligh ed in ed and blue, espec i ely. Minimum o
cumula i e GC/AT-skew (yellow) indica es he e minus o eplica ion ( e ), which is iden ical wi h he posi ion o he gene cckA. The o igin o eplica ion (o iC)
has been iden i ied be ween he genes dnaA and pa A (supplemen a y ig. S5,Supplemen a y Ma e ial online). These da a ha e been ep oduced (supple-
men a y ig.S9,Supplemen a yMa e ialonline). (B) GC-con en o he ch omosome. (C) Numbe o me hyla ed Adenosines (m6A) wi hin he GANTC mo i . (D)
Log
2
co e age o he ch omosome by RNA-seq eads o bo h s ands. Da a in A o Dha e been calcula ed o sliding windows o 4 kb. (E) Sca e plo s o DsGTA
co e age e sus GC-con en , m6A me hyla ion and log
2
RNA-seq co e age. Spea man’s qis indica ed (P<0.001). (F) Au oco ela ion unc ion showing
Pea son co ela ion calcula ed om DsGTA co e age o all pai s o sequences wi h he same dis ance om each o he so ed by inc easing dis ance. One
qua e , one hi d, and hal he ch omosomal leng h away om o iC a e indic ed by ed lines. The dashed blue line indica es he 95% con idence in e al.
Tomasch e al. GBE
366 Genome Biol. E ol. 10(1):359–369 doi:10.1093/gbe/e y005 Ad ance Access publica ion Janua y 9, 2018
Downloaded om h ps://academic.oup.com/gbe/a icle-abs ac /10/1/359/4794729
by Ges Bio echnologische use
on 07 Feb ua y 2018
2017) al hough we do no ha e di ec e idence om single
cell expe imen s wi h D. shibae.Geneexp essionisas ochas-
ic o noisy p ocess. Bis abili y can a ise om noise in he ex-
p ession o ansc ip ional egula o s embedded in posi i e o
nega i e eedback loops (Dubnau and Losick 2006). I is
emp ing o specula e ha noisy exp ession o luxI/R
2
induces
bi u ca ion o he popula ion in o cells exp essing GTA genes
and hose ha do no . Howe e , u he expe imen s will be
needed in o de o unde s and egula ion o GTA p oduc ion
in D. shibae.
Packaging o D. shibae genomic DNA in o GTA pa icles is
s ikingly di e en om ha epo ed o he o he o ganisms
o which whole genome s udies exis (Be glund e al. 2009;
Hynes e al. 2012). In hese cases mic oa ays had been used
o de e mine packaging equency o genomic DNA, a less
sensi i e me hod compa ed wi h DNA sequencing due o he
limi ed dynamic ange o he ob ained luo escence signal.
Howe e , un-o eplica ion and he esul ing highe packag-
ing equency o he ampli ied egion could be eliably
de ec ed by his me hod (Be glund e al. 2009).
Fu he mo e, ansposon-sequencing suppo ed he mic oa -
ay da a o BaGTA (Queba e e al. 2017). Thus, i is unlikely
ha he di e ences in packaging equency be ween his and
he p e ious s udies ep esen an a i ac o he di e en
me hods used.
The ques ion o exac ly how he packaging o bac e ial
DNA in o GTAs is pe o med has no been add essed ye .
Howe e , he a ailable da a would sugges a “head ul”
ype o packaging, which is common in double-s anded
DNA phages (Casjens 2011;Oli ei a e al. 2013;Black
2015). In his packaging sys em, he o en highly conca e-
me ic and b anched phage DNA is channeled in o he emp y
capsid by an enzyme complex loca ed a he capsid en y
(po al) and i is condensed 10,000- old in he p ocess
(Be ndsen e al. 2015). This biomo o , which is he as es
and mos powe ul molecula mo o known o da e (Smi h
2011), consis s o h ee essen ial componen s, each o hem
o ming oligome ic ing s uc u es: he po al p o ein, and he
la ge and small subuni s o he e minase complex, Te L and
Te S, espec i ely (Casjens 2011). Packaging is ini ia ed by
Te S binding o ecogni ion si es wi hin he conca eme ic
phage DNA. A ecen s udy sugges s ha he DNA is w apped
a ound he oligome ic Te S ing (Gao e al. 2016)andTe S-
bound DNA is hen ecognized and ini ially cu by Te L a a
de ined si e. As he nuclease domain o Te L cu s DNA unspe-
ci ically, he si e o DNA linea iza ion is de e mined by he
s uc u e o he Te S-DNA nucleop o ein complex (Djacem
e al. 2017). The ATP-dependen mo o domain o Te L
hen anspo s he linea DNA h ough he po al ing in o
he capsid. When he capsid is ull, ano he cu eleases linea
DNA ha can be packaged in o he nex capsid (Black 2015).
Te S p esen a ion o DNA owa ds Te L is indispensable o
p oduc ion and packaging o linea DNA in i o. Howe e ,
PhageT4DNApackagingisup o100%e icien in i owi h
h ee componen s only: small linea DNA o any sequence,
he la ge e minase Te L, and pu i ied p ohead wi h po al
p o eins (Black and Rao 2012). Thus, i linea DNA is p esen
in he cell, packaging could be possible e en wi hou ecog-
ni ion si es o Te S.
How hen can andom packaging o ch omosomal DNA
in o GTAs be accomplished? Genes coding o e minase and
po al p o eins a e p esen in alphap o eobac e ial GTA gene
clus e s (Lang and Bea y 2007;Tama i e al. 2017). On he
o he hand, we we e no able o iden i y sequence mo i s ha
could ac as ecogni ion si es o Te S wi hin he peaks o high
packaging. Two hypo heses migh help o explain he ob-
se ed packaging equency. Fi s , he D. shibae genome
migh encode ecogni ion si es o Te S a he co e age max-
ima ha a e so weakly conse ed ha we canno de ec
hem, o he physical p ope ies o he DNA, o example,
how easy i is o w ap he DNA a ound Te S, a e mo e im-
po an han he sequence i sel . Second, pe haps he e a e
mo e ecogni ion si es h oughou he genome, making i
di icul o iden i y hose en iched in he packaging peaks
by disc imina i e analysis. O ien a ion, h ee-dimensional
s uc u e, DNA modi ica ion and he o ma ion o nucleop o-
ein complexes o he D. shibae ch omosome and plasmids
migh hen de e mine i a egion can be accessed o pack-
aging. Many Alphap o eobac e ia, including D. shibae (Pa zel
e al. 2013), u ilize a pola mode o cell di ision. In he bes -
s udied model o ganism Caulobac e c escen us, he o igin o
eplica ion is loca ed a one cell pole, and he a ms o he
ch omosome ex end he leng h o he cell owa ds he e mi-
nus o eplica ion which is bound o he opposi e cell pole
(Bowman e al. 2008). Physical a achmen o he cell poles
migh s e ically hinde DNA o be bound by Te S, explaining
he i ually absen packaging o DNA om he ch omosomal
o igin and e minus o eplica ion. Using ch omosome con-
o ma ion cap u e and deep sequencing, he h ee-
dimensional s uc u e o he C. c escen us ch omosome was
mapped (Le e al. 2013). I showed mul iple spa ial domains
which we e s able h oughou he cell cycle; he ch omosome
con ained back-bone egions as well as so-called plec onemes
(supe -coiled s uc u es). A simila ch oma in-like s uc u e
was also ound in Bacillus sub ilis and Esche ichia coli (Dame
and Ta k-Dame 2016). The na u e o ch omosomal o ganiza-
ion in D. shibae is cu en ly unknown, bu i i is compa able
o ha o hese well-s udied model o ganisms, such o gani-
za ion in o domains ha a e dependen on localiza ion and
in e ac ion equency migh esul in some egions in which
he DNA is mo e exposed and he e o e mo e accessible o
packaging in o GTAs. This migh explain he egula dis ibu-
ion o packaging peaks h oughou he ch omosome.
Rega dless o he de ails o DNA ecogni ion by Te S, once
DNA is bound by he p o ein i would hen be cu by Te L,
esul ing in linea DNA ha can be u he packaged in o a
GTA head. As successi e packaging migh andomly abo
a e his ini ial linea iza ion, he GTA co e age would
Gene T ans e Agen s o Dino oseobac e shibae GBE
Genome Biol. E ol. 10(1):359–369 doi:10.1093/gbe/e y005 Ad ance Access publica ion Janua y 9, 2018 367
Downloaded om h ps://academic.oup.com/gbe/a icle-abs ac /10/1/359/4794729
by Ges Bio echnologische use
on 07 Feb ua y 2018