Transcriptome dynamics of the Myxococcus xanthus multicellular developmental program

*Fo co espondence:
jdo ado@ug .es
Compe ing in e es s: The
au ho s decla e ha no
compe ing in e es s exis .
Funding: See page 22
Recei ed: 20 July 2019
Accep ed: 04 Oc obe 2019
Published: 14 Oc obe 2019
Re iewing edi o : Taˆm Migno ,
CNRS-Aix Ma seille Uni e si y,
F ance
Copy igh Mun
˜oz-Do ado e
al. This a icle is dis ibu ed unde
he e ms o he C ea i e
Commons A ibu ion License,
which pe mi s un es ic ed use
and edis ibu ion p o ided ha
he o iginal au ho and sou ce a e
c edi ed.
T ansc ip ome dynamics o he
Myxococcus xan hus mul icellula
de elopmen al p og am
Jose
´Mun
˜oz-Do ado
1
*, Au elio Mo aleda-Mun
˜oz
1
,
F ancisco Ja ie Ma cos-To es
1
, F ancisco Ja ie Con e as-Mo eno
1
,
Ana Belen Ma in-Cuad ado
2
, Ja ed M Sch ade
3
, Penelope I Higgs
3
, Juana Pe
´ ez
1
1
Depa amen o de Mic obiologı
´a, Facul ad de Ciencias, Uni e sidad de G anada,
G anada, Spain;
2
Depa amen o de Fisiologı
´a, Gene´ ica y Mic obiologı
´a,
Uni e sidad de Alican e, Alican e, Spain;
3
Depa men o Biological Sciences, Wayne
S a e Uni e si y, De oi , Uni ed S a es
Abs ac The bac e ium Myxococcus xan hus exhibi s a complex mul icellula li e cycle. In he
p esence o nu ien s, cells p ey coope a i ely. Upon s a a ion, hey en e a de elopmen al cycle
whe ein cells agg ega e o p oduce mac oscopic ui ing bodies illed wi h esis an myxospo es.
We used RNA-Seq echnology o examine he ansc ip ome o he 96 h de elopmen al p og am.
These da a e ealed ha 1415 genes we e sequen ially exp essed in 10 disc e e modules, wi h
exp ession peaking du ing agg ega ion, in he ansi ion om agg ega ion o spo ula ion, o du ing
spo ula ion. Analysis o genes exp essed a each speci ic ime poin p o ided insigh s as o how
s a ing cells ob ain ene gy and p ecu so s necessa y o assembly o ui ing bodies and in o
de elopmen al p oduc ion o seconda y me aboli es. This s udy o e s he i s global iew o
de elopmen al ansc ip ional p o iles and p o ides impo an ools and esou ces o u u e
s udies.
DOI: h ps://doi.o g/10.7554/eLi e.50374.001
In oduc ion
Myxococcus xan hus is a soil-dwelling d-p o eobac e ium ha exhibi s a complex mul icellula li e
cycle wi h wo phases: g ow h and s a a ion-induced de elopmen (Mun
˜oz-Do ado e al., 2016).
When nu ien s a e a ailable, cells di ide o p oduce a communi y known as swa m. Swa ms a e
p eda o y (al hough no obliga e) and can diges p oka yo ic and euka yo ic mic oo ganisms
(Pe
´ ez e al., 2016). Upon s a a ion, cells in he swa m en e a de elopmen al p og am, du ing
which hey mig a e in o agg ega ion cen e s and climb on op o each o he o build mac oscopic
s uc u es e med ui ing bodies. To o m ui ing bodies, s a ing cells glide on solid su aces by
using wo mo ili y sys ems, known as A- (ad en u ous) and S- (social) mo ili y, which allow indi idual
cell mo emen o g oup mo emen ha equi es cell-cell con ac , espec i ely (Mau iello e al.,
2010;Nan e al., 2014;Islam and Migno , 2015;Chang e al., 2016;Schumache and Søgaa d-
Ande sen, 2017). A e comple ion o agg ega ion (24 h pos -s a a ion), cells di e en ia e in o
en i onmen ally esis an myxospo es, which a e embedded in a complex ex acellula ma ix (Fig-
u e 1). Each ui ing body con ains »10
5
–10
6
myxospo es. In e es ingly, only »10% o he s a ing
popula ion become myxospo es (O’Conno and Zusman, 1991a) as mos cells (a ound 60%)
unde go p og ammed cell dea h, mos likely o p o ide he es o he popula ion enough nu ien s
o success ully build ui ing bodies (Wi eman and Dwo kin, 1977;Na iya and Inouye, 2008). The
emaining cells di e en ia e in o a pe sis e -like s a e, e med pe iphe al ods (PR), which su ound
he ui ing bodies (O’Conno and Zusman, 1991a;O’Conno and Zusman, 1991b;O’Conno and
Mun
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TOOLS AND RESOURCES
Zusman, 1991c). While PRs a e mo phologically simila o ege a i e cells, myxospo es a e coccoid
and a e su ounded by a hick coa mainly consis ing o polysaccha ides (Ko el e al., 1975;
Mu
¨lle e al., 2010;Mu
¨lle e al., 2012;Holkenb ink e al., 2014). Myxospo es can ge mina e
when nu ien s a e a ailable, and collec i e ge mina ion o myxospo es om a ui ing body gene -
a es a small swa m ha acili a es coope a i e eeding.
The de elopmen al p og am is di ec ed by sophis ica ed, bu no comple ely de ined, gene ic
egula o y ne wo ks, which a e coupled o a se ies o in a- and ex a-cellula cues (K oos, 2017;
Mun
˜oz-Do ado e al., 2016). The i s cue is s a a ion, which igge s accumula ion o cyclic-di-
GMP and, ia he s ingen esponse, guanosine pen a- and e aphospha e [(p)ppGpp] inside he
cells. These global signals somehow ac i a e ou mas e cascade modules (Nla24, M p, F uA, and
bac e ial enhance -binding p o eins [bEBPs]), which in e connec o con ol he co ec iming o
gene exp ession (K oos, 2017). P ope p og ession o de elopmen equi es in e cellula communi-
ca ion, whe ein cells p oduce and ansmi i e sequen ial ex acellula signals, named A, B, C, D,
and E (B e l and Ki by, 2016).
Al hough much knowledge has been gene a ed in he las 40 yea s abou he M. xan hus de el-
opmen al cycle, especially wi h espec o signaling and gene egula o y ne wo ks, we a e a om
ha ing an o e all pic u e o all he e en s ha occu du ing agg ega ion and spo ula ion. Se e al
pa ial ansc ip ome analyses om de elopmen al samples based on mic oa ays ha e been pub-
lished, which we e es ic ed o a ew genes ela ed o bEBPs (Jakobsen e al., 2004;Dioda i e al.,
2008), wo-componen sys ems (TCSs) (Shi e al., 2008), A-signaling genes (Kono alo a e al.,
2012), o lipid me abolism (Bha e al., 2014). He e, we used RNA-Seq echnology o measu e
global changes in ansc ip abundance a se en ime poin s du ing M. xan hus de elopmen , which
ep esen s a subs an ial s ep o wa d compa ed o p e ious analyses. We ound ha a leas 19.6%
o M. xan hus genes (1415/7229) had s a is ically signi ican changes in ansc ip abundance du ing
de elopmen . These da a and analyses p o ide, o he i s ime, a comp ehensi e iew o he an-
sc ip ional egula o y pa e ns ha d i e he mul icellula de elopmen al p og am o his myxobac e-
ium, o e ing an essen ial sca old o u u e in es iga ions.
Resul s and discussion
T ansc ip ome analysis o he de elopmen al p og am by RNA-Seq
Global gene exp ession pa e ns we e examined by RNA-Seq analysis o he wild- ype M. xan hus
s ain DK1622 de eloped on nu ien limi ed CF aga pla es. RNA was ha es ed om wo indepen-
den biological eplica es a 0, 6, 12, 24, 48, 72, and 96 h o de elopmen , e e se ansc ibed o
cDNA, and sequenced by Illumina me hodology. On a e age, 54.72 million ead pai s and a co e -
age o 591X was ob ained. A e emo ing he ibosomal sequences, he genome co e age a ied
om 5.52 o 14.18X (median o 10.49X), enough o p o ide an adequa e co e age o he mRNA
ac ion. The wo sample- eplica es showed a high deg ee o conco dance in gene exp ession (R
2
Figu e 1. Schema ic o he M. xan hus de elopmen al p og am. The ime line indica es agg ega ion and spo ula ion phases. M. xan hus cells (yellow
ods) agg ega e in o mounds (a ows indica e gliding o agg ega ion cen e s) and hen di e en ia e in o esis an spo es (g ay ci cles) o p oduce
ma u e ui ing bodies. Pe iphe al ods (g ay ods) emain ou side o he ui ing bodies as a dis inc di e en ia ed s a e. Cells unde going lysis a e
depic ed wi h dashed lines.
DOI: h ps://doi.o g/10.7554/eLi e.50374.002
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Tools and esou ces Mic obiology and In ec ious Disease
co ela ion >0.98), wi h he excep ion o 24 h samples (R
2
co ela ion = 0.80), which may be ela ed
o lack o synch ony be ween cells in he ansi ion om agg ega ion o spo ula ion. The median o
bo h alues was u ilized o u he analysis (Table 1 and Table 1—sou ce da a 1).
As a i s da a alida ion s ep, we de e mined he exp ession p o iles o wo di e en de elop-
men al genes using b-galac osidase ansc ip ional epo e s [spiA::Tn5-lacZ (s ain DK4322) and
mgE::Tn5-lacZ (s ain DK4294)] (K oos e al., 1986) om cells de eloped unde he same condi ions
used in his s udy. Compa ison o hese b-galac osidase ac i i ies o he RNA-Seq da a indica ed he
pa e ns we e simila and in ag eemen wi h hose ob ained wi h o he s a egies (Figu e 2). Mo e-
o e , he exp ession p o iles o many genes ha ha e been p e iously cha ac e ized om b-galac o-
sidase epo e ac i i y, qRT-PCR, o mic oa ay analyses we e compa ed wi h hose ob ained wi h
ou da a. This analysis e ealed a gene al ag eemen (Figu e 2—sou ce da a 1).
Gene exp ession p o iles o ganize in o 10 de elopmen al g oups (DGs)
To iden i y de elopmen ally egula ed ansc ip s wi h simila exp ession pa e ns, genes con aining
measu ed RPKM ( eads pe kilobase pai o ansc ip pe million mapped eads) alues o all ime
poin s we e u he analyzed. Fi s , all genes wi h <50 eads and/o high eplica e a iabili y be ween
he wo eplica e da ase s (R
2
co ela ion <0.7) we e emo ed (Table 1—sou ce da a 2). 1557/7229
(21.5 %) genes passed quali y c i e ia il e s. Some o hese genes (142; 9.1%) we e no signi ican ly
(>2 old) up- o down egula ed du ing he de elopmen al p og am, sugges ing hese genes could
be conside ed cons i u i ely exp essed. 1415 signi ican ly egula ed genes (90.9% o he genes pass-
ing quali y con ol) we e hen analyzed o clus e s o simila exp ession pa e ns. B ie ly, ini ial e alu-
a ion o se e al clus e ing me hods (see Ma e ials and me hods o de ails) e ealed kmeans
clus e ing wi h 6–12 clus e s p oduced he bes clus e ing o genes wi h simila exp ession p o iles.
Re inemen o kmeans clus e s by isual inspec ion indica ed 10 DGs bes explained he numbe o
Table 1. S a is ical analysis o he M. xan hus DK1622 ansc ip ome aw da a.
Da a o each o he eplicas a 0, 6, 12, 24, 48, 72 and 96 h o de elopmen a e shown.
Sample name #Gb #mapped eads # RNA- eads #clean eads (Non- RNA) % RNA a e Co e age (x) R
2
co ela ion
WT_0_1 5.70 61906718 60713758 1192960 98.07 13.05 0.99
WT_0_2 5.62 61117544 59826984 1290560 97.89 14.12
WT_6_1 5.02 54468281 53756387 711894 98.69 7.79 1.00
WT_6_2 4.80 52436258 51759879 676379 98.71 7.40
WT_12_1 5.04 54054132 53271826 782306 98.55 8.56 0.99
WT_12_2 5.37 57646891 56798096 848795 98.53 9.29
WT_24_1 3.09 33003343 32498394 504949 98.47 5.52 0.80
WT_24_2 5.38 53962216 53018166 944050 98.25 10.33
WT_48_1 6.32 62796702 61500435 1296267 97.94 14.18 0.99
WT_48_2 5.14 34693098 34020090 673008 98.06 7.36
WT_72_1 6.43 63625946 62530758 1095188 98.28 11.98 0.99
WT_72_2 5.20 51866662 50779793 1086869 97.90 11.89
WT_96_1 6.33 63255801 62039033 1216768 98.08 13.31 0.99
WT_96_2 6.11 61187496 60080963 1106533 98.19 12.11
DOI: h ps://doi.o g/10.7554/eLi e.50374.003
The ollowing sou ce da a is a ailable o Table 1:
Sou ce da a 1. Numbe o eads o each ORF o Myxococcus xan hus a 0, 6, 12, 24, 48, 72 and 96 h o de elopmen .
DOI: h ps://doi.o g/10.7554/eLi e.50374.004
Sou ce da a 2. RPKM alues o he de elopmen al ime cou se and co ela ion sco es.
RPKM alues epo ed he e we e calcula ed om he o al numbe o non- RNA/ RNA con aining eads. The old (MXAN_) locus ags, new gene iden i-
ie s (MXAN_RS), gene name and p edic ed unc ions o pa hways in which hey ha e been p e iously implica ed a e included. The numbe o missing
da a poin s and old change we e used as c i e ia o he de elopmen al gene analysis. DG o eason ha genes we e no included in he DGs is
indica ed.
DOI: h ps://doi.o g/10.7554/eLi e.50374.005
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Tools and esou ces Mic obiology and In ec ious Disease
unique gene p o iles (Figu e 3A,Figu e 3— igu e supplemen 1, and Figu e 3—sou ce da a 1).
Clus e s we e o ganized wi h peak exp ession p o iles co esponding o p og ession h ough he
de elopmen al p og am (Figu e 3A). The ela i e exp ession p o iles p esen ed he e in he hea
maps a e a log
2
no malized RPKM alue ela i e o he mean o he en i e RNA-Seq ajec o y o a
gi en gene. This ela i e exp ession p o ile is log
2
(RPKMi ime-poin x/RPKMi a e age), whe e i is a
gi en gene, x is a gi en ime-poin , and RPKM a e age is he a e age RPKM alue o all ime-poin s.
Al hough he sensi i i y o he RNA-Seq echnology allows de ec ion o genes wi h low exp ession
le els, some genes we e excluded in his analysis a e emo ing hose wi h less han 50 eads. This
is especially impo an o genes ha a e exp essed du ing g ow h a le els no de ec able wi h his
echnology. Only 13 genes we e ound in his si ua ion (Figu e 3—sou ce da a 2), which ha e no
been included in he analyses shown below.
DGs 1 and 2 con ained genes ha we e immedia ely down- egula ed ela i e o g ow h condi-
ions, DGs 3–5 co ela ed wi h he agg ega ion phase, DGs 6–7 co ela ed wi h he ansi ion om
agg ega ion o spo ula ion, and DGs 8–10 co ela ed wi h he spo ula ion phase (Figu e 3A). The
numbe o genes a ibu ed o each clus e anged om 5.5% (DG3) o 14.1% (DG8) (Figu e 3— ig-
u e supplemen 2). Simila p opo ions o genes exhibi ed peak exp essions in g ow h (21.8%),
agg ega ion (23.8%), and ansi ion om agg ega ion o spo ula ion (18.8%) phases. The inal phase
accoun ed o peak exp essions o 35.6% o genes, consis en wi h he signi ican mo phological and
physiological ewi ing ha mus occu du ing spo e di e en ia ion and p epa a ion o ex ended
quiescence.
When he numbe o genes by RPKM alues ha accoun o 50% o all mRNA exp ession a each
ime poin was compa ed, i was obse ed ha his numbe was low (~300) du ing g ow h, and a 48
h i s eadily inc eases o 762 by he end o spo ula ion (Figu e 3— igu e supplemen 3). This sug-
ges s ha he ansc ip ome is becoming dis ibu ed ac oss a b oade numbe o genes du ing
de elopmen .
Figu e 2. Valida ion o he RNA-Seq ansc ip ion pa e ns o genes spiA (MXAN_RS20760) (A) and mgE (MXAN_RS16790) (B). b-galac osidase speci ic
ac i i y (SA) o he s ains ha bo ing lacZ usions o he espec i e genes (blue lines) compa ed o RNA-Seq RPKM alues ( ed lines) a each
de elopmen al ime poin (h). E o ba s indica e s anda d de ia ions o b-galac osidase speci ic ac i i y de e mina ion.
DOI: h ps://doi.o g/10.7554/eLi e.50374.006
The ollowing sou ce da a is a ailable o igu e 2:
Sou ce da a 1. Compa ison o exp ession p o iles o se e al de elopmen al genes desc ibed in he li e a u e and included in he DGs wi h he RPKM
p o iles om his s udy.
DOI: h ps://doi.o g/10.7554/eLi e.50374.007
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Tools and esou ces Mic obiology and In ec ious Disease
Figu e 3. The ela i e exp ession p o iles o M. xan hus genes obse ed du ing he de elopmen al p og am
compa ed o hose p e iously obse ed du ing chemical-induc ion o spo ula ion. (A) Rela i e exp ession p o iles
o signi ican ly egula ed genes a he indica ed hou s a e induc ion o s a a ion. Genes we e clus e ed in o 10
de elopmen al g oups based on he ime o peak exp ession and hen o ganized acco ding o he empo al
p og ession o de elopmen . De elopmen al g oup numbe and he phase o he de elopmen al p og am (wi h
pho og aphs o agg ega es unde he dissec ing mic oscope and cells unde he scanning elec on mic oscope)
a e indica ed o he le o he hea map. In he pho og aphs, ed, blue and yellow ba s ep esen 2 mm, 100 mm,
and 5 mm, espec i ely. (B) Rela i e exp ession le els o he genes in panel A du ing he indica ed hou s a e
chemical-induc ion o spo ula ion (Mu
¨lle e al., 2010). The posi ion o indi idual genes in panel B is ma ched o
panel A. Rela i e exp ession le els o panels A and B a e indica ed by colo code acco ding o he legend. DGs
signi ican ly ep esen ed in up-, down-, o no - egula ed spo ula ion gene se s a e indica ed by he p obabili y
alues in yellow, blue o g ay, espec i ely (C) Compa ison o up-, down-, o no - egula ed s a a ion-induced
de elopmen and chemical-induced spo ula ion gene allies. Tally o glyce ol-induced spo ula ion genes up- ( op),
down- (middle) o no - egula ed (bo om) ha a e signi ican ly en iched in he indica ed DGs.
DOI: h ps://doi.o g/10.7554/eLi e.50374.008
The ollowing sou ce da a and igu e supplemen s a e a ailable o igu e 3:
Sou ce da a 1. RPKM alues o de elopmen ally con olled genes and dis ibu ion o he genes in he en de el-
opmen al g oups.
DOI: h ps://doi.o g/10.7554/eLi e.50374.013
Sou ce da a 2. Genes wi h no eads a ime 0 h .
DOI: h ps://doi.o g/10.7554/eLi e.50374.014
Sou ce da a 3. P e iously epo ed de elopmen al genes and iden i ica ion in he 10 de elopmen al g oups
shown in Figu e 3A.
DOI: h ps://doi.o g/10.7554/eLi e.50374.015
Sou ce da a 4. Genes in ol ed in de elopmen ha a e no included in he DGs shown in Figu e 3A and easons
o hei exclusion.
DOI: h ps://doi.o g/10.7554/eLi e.50374.016
Sou ce da a 5. Genes included o compa ison o de elopmen al and spo ula ion ansc ip omes (Figu e 3B).
DOI: h ps://doi.o g/10.7554/eLi e.50374.017
Sou ce da a 6. Tally o en ichmen o spo ula ion ansc ip ome genes (Mu
¨lle e al., 2010) ound in each o he
DGs (Figu e 3B).
DOI: h ps://doi.o g/10.7554/eLi e.50374.018
Sou ce da a 7. Tally o genes obse ed in bo h he de elopmen al ( his s udy) and spo ula ion [Mu
¨lle e al.,
2010 BMC Genomics 11:264] ansc ip omes as suppo ing da a o Figu e 3C.
DOI: h ps://doi.o g/10.7554/eLi e.50374.019
Figu e 3 con inued on nex page
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Tools and esou ces Mic obiology and In ec ious Disease

As a i s s ep in analysis o he DGs, genes ha ha e been p e iously desc ibed o a ec M. xan-
hus de elopmen we e iden i ied. O he ~280 cha ac e ized genes, 167 we e included in he DGs.
Mos showed peak exp ession pa e ns a a ime poin ha ma ched wi h he de elopmen al phase
whe e hey ha e been epo ed o unc ion, some o which ha e a well-de ined ole on de elopmen
(Figu e 3—sou ce da a 3 and Figu e 3— igu e supplemen 4). A no able excep ion o his a e
genes ha a e hough o unc ion in e y ea ly s ages du ing de elopmen , bu a e included in
DG10. Examples o hese genes a e sasS, which is in ol ed in A signaling (Yang and Kaplan, 1997);
csgA, which encodes he C signal (Shimke s and Ra iee, 1990); and omR, which is in ol ed in
pola i y con ol o mo ili y (Leona dy e al., 2007). Howe e , DG10 genes also showed high exp es-
sion a 6 h , consis en wi h ea ly ac i i ies and aising he possibili y o addi ional unc ions a he
la es s age o de elopmen . Finally, no all known de elopmen al genes appea ed in he 10 DGs
de ined he e (Figu e 3—sou ce da a 4). O he 108 missing genes, 28 genes encoded TCSs and 20
se ine/ h eonine p o ein kinases (STPKs).
Genes iden i ied h ough chemical induc ion o spo ula ion mainly map
o DG7 and DG8
A co e spo ula ion ansc ip ome was p e iously de ined using an a i icial me hod o inducing
spo e di e en ia ion (Mu
¨lle e al., 2010). Using his me hod, myxospo es can be induced in cells
g owing in ich b o h cul u e by addi ion o chemicals such as 0.5 M glyce ol (Dwo kin and Gibson,
1964). Chemical-induced spo ula ion bypasses he equi emen o s a a ion, mo ili y (agg ega ion),
and al e na e cell a es (Higgs e al., 2014). Compa ison o hese wo ansc ip ome se s de e -
mined ha 1388 genes passed quali y c i e ia in bo h ansc ip ome s udies (Figu e 3B,Figu e 3—
sou ce da as 5 and 6,Figu e 3C, and Figu e 3—sou ce da a 7). 92% (218/237) o genes signi i-
can ly up- egula ed (>2 old) in he spo ula ion ansc ip ome we e also signi ican ly up- egula ed in
he de elopmen al ansc ip ome. These genes we e signi ican ly o e - ep esen ed in DGs 7 and 8,
wi h low p obabili y (p) ha his associa ion is due o andom chance (p=110
12
and p=210
11
,
espec i ely). DGs 7 and 8 peak exp ession a he ansi ion o spo ula ion and spo ula ion phases,
espec i ely. Co- egula ed genes in hese g oups include hose p edic ed o be in ol ed in gene a-
ion o suga p ecu so s o spo e coa (see below). Likewise, gene ic loci in ol ed in spo e coa syn-
hesis (exo) and su ace polysaccha ide a angemen (n s) (Mu
¨lle e al., 2012;Holkenb ink e al.,
2014) ell in DG8.
In e es ingly, DGs 9 and 10, wi h peak exp ession p o iles co esponding o he la es de elop-
men al ime poin s, we e no well ep esen ed in he up- egula ed spo ula ion ansc ip ome. DG9
genes we e signi ican ly o e - ep esen ed in he down- egula ed spo ula ion ansc ip ome
(p=0007). The DG9 clus e ep esen s genes ha we e exp essed du ing g ow h in ich media,
down- egula ed in esponse o s a a ion, and la e up- egula ed du ing he inal phases o de elop-
men (Figu e 3A). Genes in his clus e appea o be in ol ed in anspo , espi a ion, and ansc ip-
ional egula ion, and may be equi ed du ing apid g ow h and pe haps in he inal ma u a ion
phases o spo ula ion. I is likely ha he chemical-induced spo ula ion ansc ip ome may no ha e
included e y la e induced spo ula ion genes as RNA was ha es ed un il ou -hou s a e induc ion.
Chemical-induced spo es a e hea and sonica ion esis an a his s age, bu inal ma u a ion may
con inue pas his poin . Finally, DG10 con ained genes ha we e o e - ep esen ed (p=710
10
) in
Figu e 3 con inued
Figu e supplemen 1. Rela i e exp ession p o iles o signi ican ly egula ed genes a he indica ed hou s a e
induc ion o s a a ion.
DOI: h ps://doi.o g/10.7554/eLi e.50374.009
Figu e supplemen 2. Tally o eliable genes.
DOI: h ps://doi.o g/10.7554/eLi e.50374.010
Figu e supplemen 3. Numbe o genes composing 50% o he ansc ip ome h oughou he de elopmen al
p og am.
DOI: h ps://doi.o g/10.7554/eLi e.50374.011
Figu e supplemen 4. A: Rela ionship be ween M. xan hus de elopmen al phases, de elopmen al g oups, and a
known unc ional in e ac ion ne wo k.
DOI: h ps://doi.o g/10.7554/eLi e.50374.012
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Tools and esou ces Mic obiology and In ec ious Disease
he no - egula ed spo ula ion ansc ip ome gene se . We specula e his pool o genes may be p es-
en in PRs, a cell a e en iched la e in he de elopmen al p og am and no p esen du ing chemical
induc ion o spo ula ion. Genes in his DG encode se e al STPKs, he bEBP Nla26, and se e al p o-
eins in ol ed in seconda y me aboli e (SM) biosyn hesis (see below).
Good co ela ion was obse ed in genes ha we e exp essed du ing g ow h and down- egula ed
in bo h ansc ip ome se s, wi h genes om DGs 1, 2, 5, and nine being o e - ep esen ed in he
down- egula ed spo ula ion ansc ip ome se (Figu e 3C). O he la ge pool o genes ha we e no
signi ican ly egula ed in he spo ula ion ansc ip ome (754), a ela i ely small numbe (12%) we e
also no signi ican ly up- o down- egula ed in he de elopmen al ansc ip ome. This pool o genes
likely ep esen s cons i u i ely exp essed genes ha se e as good no maliza ion ma ke s (Figu e 3—
sou ce da a 5) and includes housekeeping genes such as he ansc ip ion e mina ion ac o Rho
(MXAN_RS11995), he cell cy oskele al p o ein M eB (MXAN_RS32880) (Mu
¨lle e al., 2012;T eu-
ne -Lange e al., 2015;Fu e al., 2018), he gliding mo ili y and spo ula ion p o ein AglU
(MXAN_RS14565) (Whi e and Ha zell, 2000), and he CheA homolog Di E (MXAN_RS32400), which
is equi ed o exopolysaccha ide p oduc ion and social mo ili y (Yang e al., 2000).
Analysis o de elopmen al egula ed genes
All genes included in he 10 DGs we e indi idually analyzed o ind ou which p ocesses we e
a ec ed du ing de elopmen , which may explain he di e en e en s ha occu du ing agg ega ion
and spo ula ion. He e, we ha e ocused on six di e en p ocesses.
A- and S-mo ili y genes exhibi di e en de elopmen al exp ession p o iles
The wo phases o he M. xan hus li ecycle (p eda o y g ow h and de elopmen ) depend on A- and
S-mo ili y engines and hei associa ed egula o y p o eins (Pe
´ ez e al., 2014;Islam and Migno ,
2015;Me cie and Migno , 2016;Schumache and Søgaa d-Ande sen, 2017). Ou da a ha e
e ealed ha many o he mo ili y genes we e de elopmen ally egula ed and ha he exp ession
p o iles o he dis inc A- and S-machine y genes clea ly di e ed du ing de elopmen (Figu e 4).
A-mo ili y genes we e up- egula ed du ing ea ly de elopmen , while S-mo ili y genes i s dec eased
a 6 h , and hen e u ned o g ow h le els du ing agg ega ion (excep o pilA), sugges ing ha A
mo ili y is p e e en ially used by cells du ing agg ega ion. Du ing spo ula ion, exp ession o genes
encoding bo h mo ili y engines dec eased (Figu e 4), al hough some o hem peaked again a his
s age. The peak o some A-mo ili y genes du ing spo ula ion is consis en wi h he epu posing o
ce ain A-mo ili y p o eins o unc ion in spo e coa assembly (Wa el e al., 2013). A he end o
de elopmen , he exp ession le els o he mo ili y genes emain high. Howe e , i should be
eminded ha al hough ma u e ui ing bodies a e s a ic s uc u es, he PRs su ounding hem a e
mo ile (O’Conno and Zusman, 1991b).
Gene exp ession pa e ns a e consis en wi h use o glycogen and lipid
bodies as ene gy sou ces
Du ing g ow h, M. xan hus does no appea o consume suga s as ca bon o ene gy sou ces
(Wa son and Dwo kin, 1968;B e sche and Kaise , 1978). Ins ead, py u a e, amino acids, and lip-
ids a e e icien ly u ilized which di ec ly en e he ica boxylic acid (TCA) cycle (B e sche and Kai-
se , 1978). I has been long deba ed as o whe he M. xan hus u ilizes a ully unc ioning glycoly ic
pa hway (Cu is and Shimke s, 2008). I was specula ed ha he pa hway may be u ilized p ima ily
in he gluconeogenic di ec ion o p oduce suga p ecu so s necessa y o spo e coa p oduc ion
(Youde ian e al., 1999;Cha i a e al., 2007;Ge sin e al., 2013). I is unknown how hese pa h-
ways con ibu e o ene gy p oduc ion du ing de elopmen when s a ing cells mus syn hesize
ene gy cu encies (i.e. ATP) o e a pe iod o a leas h ee days.
Analysis o he DGs e ealed ha mos genes in ol ed in ene gy gene a ion (py u a e dehyd o-
genase complex, TCA cycle and oxida i e phospho yla ion) we e ound in DG2 (Figu e 5A). The e-
o e, al hough hey a e down- egula ed a 6 h , hey a e la e up- egula ed a lowe le els han
du ing g ow h. In con as , many genes encoding enzymes o he glycoly ic/gluconeogenic pa hway
we e up- egula ed du ing de elopmen , wi h mos eaching maximum exp ession le els a he com-
ple ion o agg ega ion (24–48 h ) (Figu e 5B). This up- egula ed g oup included genes encoding
homologs o glucokinase (glkC) and phospho uc okinase (p kA), which a e speci ic o he glycoly ic
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Tools and esou ces Mic obiology and In ec ious Disease
Figu e 4. De elopmen al exp ession le els o M. xan hus mo ili y p o eins. Schema ic ep esen a ion o he ocal adhesion mo o complexes necessa y
o A mo ili y ( op), he ype IV pili mo o complexes necessa y o S mo ili y (bo om), and he p o eins in ol ed in con olling pola i y o bo h engines
(pola i y con ol module; cen e ). The de elopmen al exp ession le els (RPKM) o signi ican ly egula ed mo ili y genes a he indica ed imes (h) o
Figu e 4 con inued on nex page
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Tools and esou ces Mic obiology and In ec ious Disease
pa hway. These obse a ions a e consis en wi h a ansc ip ional ewi ing o me abolic pa hways
du ing he de elopmen al p og am, pe haps o ake ad an age o changing ca bon/ene gy sou ces.
Fo ins ance, de elopmen al up- egula ion o he glycoly ic pa hway genes may allow de eloping
cells o ob ain ene gy om suga s eleased om cells unde going de elopmen al lysis o om glyco-
gen. Glycogen accumula es du ing la e s a iona y phase/ea ly de elopmen , and hen disappea s
p io o spo ula ion (Na iya and Inouye, 2003). Enzymes p edic ed o be in ol ed in syn hesis o
glycogen, such as GlgC, we e ound in DG4, while enzymes in ol ed in u iliza ion o glycogen, such
as ehalose syn hase, GlgP, and MalQ appea ed o be cons i u i ely exp essed. The obse a ion
ha hese compe ing pa hways show o e lapping exp ession p o iles sugges s ha egula ion o
glycogen p oduc ion/consump ion is likely egula ed pos - ansc ip ionally, as has been demon-
s a ed by phospho yla ion o P kA by Pkn4 (Na iya and Inouye, 2003).
Lipid bodies also accumula e in cells p io o spo ula ion. They mainly consis o iacylglyce ides
de i ed om memb ane phospholipids as cells sho en in leng h, which a e la e used as an ene gy
sou ce (Hoiczyk e al., 2009;Bha e al., 2014). Howe e , he p o iles o genes in ol ed in bo h
s aigh - and b anched-chain p ime syn hesis and elonga ion o a y acids obse ed in his s udy
(Figu e 6A and B) sugges ha some le el o lipid syn hesis occu s du ing de elopmen . Mo eo e ,
genes in ol ed in he al e na i e pa hway o p oduce iso ale yl-CoA (Bode e al., 2009) we e
induced (Figu e 6A and B). These lipids migh be ei he inco po a ed in o lipid bodies, be esponsi-
ble o changes in lipid composi ion o he memb anes o myxospo es and/o PRs, and/o be used
as p ecu so s o SMs p oduc ion. On he o he hand, genes in ol ed in lipid deg ada ion eached
maximum exp ession a 24–48 h (b-oxida ion) o e en a la e imes (by o he pa hways)
(Figu e 6C). These a y acid deg ada ion enzyme p o iles sugges ha lipids a e p e e ably con-
sumed du ing spo ula ion.
These da a o e an o e all pic u e o he cen al me abolism du ing de elopmen , which ein o -
ces he no ion ha mac omolecules ecycled om g ow h phase o eleased om lysing cells can be
di ec ly used o yield ene gy, bu a e also used o syn hesize glycogen and lipids ha a e s o ed o
la e consump ion.
Amino acid and suga p ecu so s equi ed o de elopmen al
mac omolecule syn hesis may be eleased by p o ein and polysaccha ide
u no e and gluconeogenesis
In addi ion o ene gy, s a ing cells need a sou ce o suga p ecu so s o syn hesize de elopmen ally
speci ic polysaccha ides equi ed o mo ili y (Li e al., 2003), ui ing body encasemen (Lux and
Shi, 2005), spo e coa syn hesis (Ko el e al., 1975;Holkenb ink e al., 2014), and spo e esis-
ance (McB ide and Zusman, 1989). I has been sugges ed ha hese suga s a e de i ed om glu-
coneogenesis (Youde ian e al., 1999). Consis en ly, ou da a ha e e ealed ha genes encoding
enzymes speci ic o gluconeogenesis, such as phosphoenolpy u a e ca boxykinase was in DG2, and
GlpX ( uc ose-1,6-bisphospha ase) we e p esen du ing g ow h and h oughou de elopmen
(Table 1—sou ce da a 2). Thus, hese obse a ions, as well as hose p esen ed abo e (Figu e 5B),
indica e ha gluconeogenesis likely con ibu es o suga p ecu so p oduc ion a a ious s ages du -
ing de elopmen . Mo eo e , he obse a ion ha ou glycosyl hyd olases we e speci ically up- egu-
la ed du ing de elopmen (Figu e 7A) sugges s he cells may ecycle ege a i e polysaccha ides o
sca enge polysaccha ides eleased om cells induced o lyse. These eleased ee monome s could
be syn hesized in o speci ic de elopmen al polysaccha ides by he se ies o glycosyl ans e ases
ha a e also de elopmen ally up- egula ed (Figu e 7B).
The de elopmen al p og am is mainly igge ed by amino acid s a a ion, ye de elopmen ally
speci ic p o eins need o be newly syn hesized. A sou ce o hese amino acids could be eleased
om lysed cells and u no e o p o eins ha a e no equi ed du ing de elopmen . Consis en ly, 60
p o eases appea ed in he 10 DGs. 16 o hese we e speci ic o g ow h and down- egula ed a e 6
Figu e 4 con inued
de elopmen a e depic ed. Gene exp ession p o iles a e colo ed o ma ch he p o eins shown in he schema ic. P o eins depic ed in g ay ep esen
genes ha we e no included in he de elopmen al g oups.
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Tools and esou ces Mic obiology and In ec ious Disease
showed exp ession pa e ns ha sugges ed hey play impo an oles in modula ion o agg ega ion
and/o spo ula ion.
21 OCS genes we e de elopmen ally egula ed, 9 o which we e down- egula ed. The emaining
we e di e en ially up- egula ed du ing de elopmen (Figu e 10A), including he ep esso LexA
(Campoy e al., 2003), and he egula o s SasN (Xu e al., 1998) and M pC (Sun and Shi, 2001;
Ueki and Inouye, 2003;McLaughlin e al., 2018).
O he 272 encoded TCS genes, we ound 47 included in he DGs: 23 HKs, 10 hyb id HKs (HyHKs,
con aining HK and RR modules in he same polypep ide), and 14 RRs. 6 TCS genes we e shu down
du ing de elopmen (Figu e 10B). Only 16 o 47 TCS genes ha e been p e iously cha ac e ized,
hus pinpoin ing addi ional candida es o u he cha ac e iza ion. Addi ionally, 5 o he 14 RRs
belong o he g oup o bEBPs (plus MXAN_RS07605, which exhibi s an a chi ec u e simila o bEBPs,
bu con ains a GAF ins ead o a ecei e domain). bEBPs unc ion o ac i a e exp ession om sigma
54 dependen p omo e s (Mo e and Sego ia, 1993). Besides he bEBPs, F uA is he only o he RR
ound in he DGs ha con ains a DNA-binding domain. Ou o he emaining 8 RRs, i e we e s and-
alone ecei e domains (CheY-like), wo con ain a pu a i e diguanyla e cyclase ou pu domain, one
o which has been cha ac e ized (Sko nicka e al., 2016), and one is RomR, which modula es mo ili y
(Leona dy e al., 2007). As many o he HKs and RRs ha we e de elopmen ally egula ed a e
o phans, he esul s p esen ed he e may help o iden i y cogna e pai s.
Rega ding o he ansc ip ion ac o s, nine sigma ac o genes we e ound o be de elopmen ally
egula ed in a sequen ial ashion (Figu e 10C), including hose encoding he majo sigma ac o
RpoD (Inouye, 1990) and RpoN (sigma 54) (Kesele and Kaise , 1997). The exp ession p o iles o
hese wo sigma ac o genes clea ly di e du ing de elopmen . While poD was down- egula ed a
6 h , up- egula ed du ing agg ega ion, and hen down- egula ed du ing spo ula ion, poN is up- eg-
ula ed h oughou de elopmen (Figu e 10D). The exp ession p o iles o poN and bEBP genes a e
consis en wi h p e ious esul s demons a ing ha a bEBP cascade is igge ed upon s a a ion
(Giglio e al., 2011). Addi ionally, sigC, which encodes a g oup II sigma ac o (Apelian and Inouye,
1993), was iden i ied in DG7. The emaining six a e p edic ed o encode ex acy oplasmic unc ions
(ECF) sigma ac o s, including poE1, in ol ed in mo ili y (Wa d e al., 1998). I is no ewo hy ha
he genes encoding he ou subuni s o he co e RNA polyme ase a e de elopmen ally egula ed
wi h p o iles simila o ha obse ed o he ibosomal p o eins (Figu e 10E).
M. xan hus encodes 99 STPKs, 11 o which ha e been epo ed o be pseudokinases, because
hey lack a leas one o he h ee equi ed ca aly ic esidues (Mun
˜oz-Do ado e al., 1991;
Pe
´ ez e al., 2008). 22 STPK genes we e included in he DGs (Figu e 10F). In e es ingly, se en
encode pseudokinases while 15 encode p edic ed ac i e kinases (Figu e 3—sou ce da as 1 and 3).
Toge he , hese obse a ions sugges ha a high numbe o egula o s di ec s he de elopmen al
p og am o M. xan hus, wi h some ac ing simul aneously and o he s sequen ially o pe ec ly modu-
la e he di e en e en s ha occu h ough de elopmen . In o he model bac e ia such as Caulo-
bac e c escen us, mo e han 19% o he genes ha e been ound o be de elopmen ally egula ed
(Laub e al., 2000), a simila pe cen age o ha ound in M. xan hus, 57% o which a e unde di ec
con ol o he i e mas e egula o s iden i ied in his bac e ium (Zhou e al., 2015). In Bacillus sub i-
lis, ou o 4100 genes, 520 we e dependen on Spo0A, bu no on s
F
, while 66 we e dependen
upon bo h egula o y p o eins (Fawce e al., 2000). And in he case o S ep omyces coelicolo ,
1901 genes (24% o he ORFs) exhibi di e ences when he subs a e mycelium di e en ia es o a
mul inuclea ed mycelium, wi h a la ge numbe o ansc ip ional egula o s in ol ed (Yagu
¨e e al.,
2013). Al hough he numbe o egula o s included in he DGs is la ge in M. xan hus han in o he
model bac e ia, i emains o be elucida ed how many o hem di ec ly modula e de elopmen .
Figu e 9 con inued
was omi ed om he locus ag o each gene. (C) De elopmen al exp ession le els (RPKM) o he pa alogous genes encoding p o ein S4 plo ed
agains de elopmen al ime poin s in hou s.
DOI: h ps://doi.o g/10.7554/eLi e.50374.025
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Tools and esou ces Mic obiology and In ec ious Disease

Figu e 10. De elopmen al exp ession p o iles o genes in ol ed in ansc ip ional egula ion and signal ansduc ion. Rela i e exp ession le els o
genes encoding one-componen egula o s (A), wo-componen signal ansduc ion p o eins (B), sigma ac o s (C), and se ine/ h eonine p o ein kinases
(F) a e depic ed. Rela i e exp ession le els o panels A, B, C, and F a e indica ed by colo code acco ding o he legend a he bo om, and
de elopmen al ime poin s in hou s a e indica ed abo e each panel. The MXAN_RS designa ion was omi ed om he locus ag o each gene.
Figu e 10 con inued on nex page
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Tools and esou ces Mic obiology and In ec ious Disease
Towa d elucida ion o a comple e de elopmen al gene- egula o y
ci cui
M. xan hus is an ex ao dina y bac e ium wi h a la ge coding po en ial and a complex li ecycle. The
de elopmen al cycle consis s o wo consecu i e e en s: agg ega ion and spo ula ion, du ing which
cells seg ega e in o h ee di e en cell a es. Mo eo e , his p ocess las s o e h ee days and is ig-
ge ed by s a a ion. Du ing his ex ended pe iod, s a ing cells mus glide o build ui ing bodies
and syn hesize nume ous mac omolecules exclusi e o ui ing bodies and myxospo es. Consis en
wi h his complexi y, he ansc ip omic analyses p esen ed he e e ealed ha 1415 genes a e de el-
opmen ally egula ed wi h a high deg ee o con idence, exhibi ing exp ession imes ha peak a
ei he g ow h, agg ega ion and/o spo ula ion. Analysis o indi idual genes and he p ocesses in
which hey pa icipa e has shed some ligh abou how cells egula e he exp ession o mo ili y genes
o allow he cells o each he agg ega ion cen e s, o how hey ewi e me abolism o bo h ob ain
ene gy and monome s o build new mac omolecules o o syn hesize SMs. Mo eo e , hese da a
ha e also e ealed ha he ansla ional and ansc ip ional machine y is deeply al e ed o modula e
he di e en e en s o de elopmen , o e ing new insigh s ha equi e o be expe imen ally pu sued
o de e mine he unc ion o all hese egula o s.
Al hough he ole o ansla ion in he egula ion o he de elopmen al cycle has no ye been
add essed, a la ge numbe o ansc ip ional egula o s ha unc ion du ing de elopmen ha e been
iden i ied (Figu e 3— igu e supplemen 4, and Figu e 11—sou ce da a 1). The da a p esen ed
he e co obo a e he esul s ob ained by o he myxobac e iologis s. Fo ins ance, ou analyses a e in
good ag eemen wi h an es ablished model in which s a a ion ac i a es ou gene ic egula o y ne -
wo ks: bEBP, M p, F uA, and Nla24 modules, wi h he la e being ac i a ed by cyclic-di-GMP
(K oos, 2017). Howe e , he high numbe o ansc ip ional egula o s and signaling p o eins ound
in his s udy o be de elopmen ally egula ed a he mRNA le el is much highe han expec ed. As
shown in Figu e 11, many uncha ac e ized egula o s peak a ei he agg ega ion o spo ula ion (Fig-
u e 11—sou ce da a 1). Those ha a e exp essed a he same ime poin may be in e connec ed o
p ope ly modula e speci ic e en s and gua an ee he p ope sequen ial exp ession o genes. The
analysis o he exp ession le els o he di e en egula o s has e ealed ha uA and m pC exhibi
maximum RPKM alues o 7046 and 4415, espec i ely, while none o he o he s each 1000 (excep
o wo sigma ac o s) (Figu e 11—sou ce da a 1). This may explain why F uA and M pC a e consid-
e ed mas e egula o s o de elopmen , while many o he es ha e no been iden i ied as playing
c ucial oles in he li ecycle o M. xan hus. Undoub edly, he de elopmen al mRNA exp ession p o il-
ing p esen ed he e will ac as a bluep in o he comple e elucida ion o he M. xan hus de elop-
men al egula o y p og am. Now ha he changes in gene exp ession a e measu ed, iden i ying he
egula o y inpu s o each p omo e will be c i ical o unde s and he comple e gene ic ci cui y con-
olling de elopmen .
Ma e ials and me hods
Key esou ces able
Reagen ype (species)
o esou ce Designa ion Sou ce o e e ence Iden i ie s Addi ional in o ma ion
S ain, s ain
backg ound
(Myxococcus xan hus)
DK1622 Kaise , 1979;
Goldman e al., 2006
Genome:
NC_008095.1
Wild- ype s ain used
o ob ain RNA
S ain, s ain
backg ound
(Myxococcus xan hus)
DK4322 K oos e al., 1986 MXAN_RS20760;
MXAN_4276
spiA::Tn5-lacZ;
Tn5 lac (Km
)W4521
Con inued on nex page
Figu e 10 con inued
Exp ession le els (RPKM) o genes encoding he majo sigma ac o s (D) and he subuni s o he RNA polyme ase (E) plo ed agains de elopmen al
ime poin s in hou s.
DOI: h ps://doi.o g/10.7554/eLi e.50374.026
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Tools and esou ces Mic obiology and In ec ious Disease
Con inued
Reagen ype (species)
o esou ce Designa ion Sou ce o e e ence Iden i ie s Addi ional in o ma ion
S ain, s ain
backg ound
(Myxococcus xan hus)
DK4294 K oos e al., 1986 MXAN_RS16790;
MXAN_ 3464
mgE::Tn5-lacZ;
Tn5 lac (Km
)W4406
Chemical
compound, d ug
DNase I Sigma-Ald ich Ca No./ID: AMPD1
Chemical
compound, d ug
Supe Sc ip III
Re e se
T ansc ip ase
In i ogen Ca No./ID: 18080044
Chemical
compound, d ug
Esche ichia coli
DNA polyme ase
New England
Biolabs
D1806
Chemical
compound, d ug
E. coli RNAse H In i ogen Ca No./ID: 18021071
Chemical
compound, d ug
P o einase K Ambion Ca No./ID: 25530–015
Chemical
compound, d ug
E. coli DNA ligase New England
Biolabs
Ca No./ID: M0205L
Chemical
compound, d ug
lysozyme Roche Ca No./ID: 10837059001
Comme cial
assay o ki
RNeasy Midi Ki Qiagen Ca No./ID: 75142
Comme cial
assay o ki
RNA P o ec
Bac e ia Reagen
Qiagen Ca No./ID: 76506
So wa e, algo i hm BWA so wa e Li and Du bin, 2009
So wa e, algo i hm SAM ools Li e al., 2009
So wa e, algo i hm A emis .16.0.0 Ru he o d e al., 2000
So wa e, algo i hm Clus e 3
So wa e Package
de Hoon e al., 2004
P epa a ion o cells o RNA-Seq expe imen
M. xan hus s ain DK1622 (Kaise , 1979;Goldman e al., 2006) was used in his s udy. Cells we e
g own in CTT liquid medium (Hodgkin and Kaise , 1977) a 30˚C wi h igo ous shaking (300 pm) o
3.0 10
8
cells/ml (op ical densi y a 600 nm [OD
600
] o 1), and hen ha es ed and esuspended in
TM bu e (10 mM T is-HCl [pH 7.6]; 1 mM MgSO
4
) o a calcula ed densi y o 4.5 10
9
cells/ml
(OD
600
o 15). Fo each ime eplica e, 200 ml aliquo s o concen a ed cell suspension we e spo ed
on o hi een sepa a e CF aga pla es (Hagen e al., 1978). Two eplica es o cells we e ha es ed
om pla es a 6, 12, 24, 48, 72 and 96 h (samples WT_6, WT_12, WT_24, WT_48, WT_72 and
WT_96, espec i ely), and he ob ained pelle s we e ans e ed immedia ely in o 0.5 ml o RNA P o-
ec Bac e ia Reagen (Qiagen). Cells we e hen incuba ed a oom empe a u e o 5 min, ha es ed
by cen i uga ion a 5000 g o 10 min (4˚C), and s o ed a 80˚C a e emo al o he supe na an .
Fo he = 0 samples (sample WT_0), wo eplica es o 30 ml o he o iginal liquid cul u e (OD
600
o
1) we e ha es ed by cen i uga ion as abo e, esuspended in RNA P o ec Bac e ia Reagen , and
p ocessed in he same manne .
RNA ex ac ion
To isola e RNA, ozen pelle s we e hawed and esuspended in 1 ml o 3 mg/ml lysozyme (Roche)
and 0.4 mg/ml p o einase K (Ambion) p epa ed in TE bu e (10 mM T is-HCl; 1 mM e hylenediami-
ne e aace ic acid [EDTA], pH 8.0) o cell lysis. Samples we e incuba ed 10 min a oom empe a-
u e. RNA ex ac ion was ca ied ou using he RNeasy Midi Ki (Qiagen) and each sample was
elu ed in 300 ml o RNase- ee wa e . The concen a ion o RNA was measu ed using a NanoD op
ND-2000 spec opho ome e (NanoD op Technologies, USA). To emo e DNA, each RNA sample
was supplemen ed wi h 1 uni o DNase I ( om he DNA Ampli ica ion G ade Ki o Sigma) pe mg
o RNA and incuba ed a oom empe a u e o 10 min. The eac ion was s opped by adding he
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Tools and esou ces Mic obiology and In ec ious Disease
s op solu ion included in he ki and incuba ing 10 min a 70˚C. The ob ained RNA was p ecipi a ed
wi h 1/10 ol o 3 M sodium ace a e and 3 olumes o e hanol, and esuspended in 50 ml o RNase-
ee wa e . The quali y o he o al RNA was e i ied by aga ose gel elec opho esis, and he concen-
a ion was de e mined using NanoD op as indica ed abo e.
Double s anded copy DNA syn hesis
Fi s s and DNA was syn hesized using Supe Sc ip III Re e se T ansc ip ase (In i ogen) s a ing
wi h 5 mg o RNA in a inal eac ion olume o 20 ml. In he nex s ep, second-s and DNA was syn-
hesized by adding 40 uni s o Esche ichia coli DNA polyme ase (New England Biolabs), 5 uni s o E.
coli RNAse H (In i ogen), 10 uni s o E. coli DNA ligase (New England Biolabs), 0.05 mM ( inal con-
cen a ion) o dNTP mix, 10x second-s and bu e (New England Biolabs), and wa e o 150 ml. A e
2 h a 16˚C, he eac ion was s opped wi h 0.03 mM EDTA ( inal concen a ion). The ob ained DNA
was pu i ied and concen a ed using he DNA Clean and Concen a o Ki o Zymo Resea ch acco d-
ing o manu ac u e ’s ins uc ions. The inal p oduc was elu ed in DNA elu ion bu e om he ki o
each, a leas , a yield o 2 mg o DNA, wi h a minimum concen a ion o 200 ng/ml.
Figu e 11. New signaling p o eins ha a e de elopmen ally egula ed in M. xan hus iden i ied in his s udy. Code used o dis inguish among ypes o
egula o s is indica ed in he uppe pa , whe e OCS indica es one-componen sys ems; HK, his idine kinase: hyHK, hyb id his idine kinase; CheY, CheY-
like esponse egula o ; RR, esponse egula o ; ECF, ECF sigma ac o ; STPK, ac i e Se /Th p o ein kinase; PseudoK, pseudokinase. Numbe s inside
each symbol indica e he numbe o each ype o egula o ha ha e no been p e iously iden i ied as being de elopmen ally egula ed. In o ma ion
abou p o eins depic ed he e is shown in Figu e 11—sou ce da a 1.
DOI: h ps://doi.o g/10.7554/eLi e.50374.027
The ollowing sou ce da a is a ailable o igu e 11:
Sou ce da a 1. Regula o y elemen s included in he de elopmen al g oups.
DOI: h ps://doi.o g/10.7554/eLi e.50374.028
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Tools and esou ces Mic obiology and In ec ious Disease
Sequencing and ansc ip omic da a analysis
The cDNA om wo biological eplica es o each condi ion (see abo e) was used o sequencing
using he Illumina HiSeq2000 (100 bp pai ed-end ead) sequencing pla o m (GATC Bio ech, Ge -
many). Sequence eads we e p e-p ocessed o emo e low-quali y bases. Nex , eads we e mapped
agains M. xan hus DK1622 ibosomal ope on sequences using BWA so wa e wi h he de aul
pa ame e s (Li and Du bin, 2009). Remaining eads we e subsequen ly mapped o he genome
sequence wi h he de aul pa ame e s and using he pai -end s a egy. SAM ools (Li e al., 2009)
was used o con e esul ing da a in o BAM o ma . A emis .16.0.0 (Ru he o d e al., 2000) was
used o he isualiza ion o he sequence eads agains he M. xan hus genome. Once he an-
sc ip s we e mapped o he genome, he a e age median alue o each condi ion was used in u -
he analyses (Table 1 and Table 1—sou ce da a 1 and 2).
De elopmen al gene analysis
Genes wi h ewe han 50 eads in a gi en ime poin we e emo ed om analysis. RPKM alues o
he emaining genes we e hen compa ed ac oss he de elopmen al ime-poin s. De elopmen al
exp ession was cha ac e ized by a > 2 old change in RPKM alues ac oss he ime cou se and
a > 0.7 R
2
co ela ion coe icien o he wo ime cou se eplica es. Log
2
old-change calcula ions
we e pe o med using he Clus e 3 So wa e Package (de Hoon e al., 2004). Genes passing hese
c i e ia a e p esen ed in Figu e 3—sou ce da a 1. Randomiza ion o he ime poin RPKM alues
wi hin each eplica e da a se yielded a alse disco e y a e o 3.97% based on i e andomized simu-
la ions ha sc ambled he o de o he ime poin s ac oss all genes in he wo da ase s. Genes pass-
ing he de elopmen al exp ession c i e ia we e hie a chically clus e ed using pea son co ela ion,
spea man ank, euclidian dis ance, and kmeans clus e ing. By isually inspec ing he clus e s we
ound ha kmeans clus e ing ga e he bes clus e ing o genes wi h simila exp ession p o iles.
Assay o b-galac osidase ac i i y
Fo quan i a i e de e mina ion o b-galac osidase ac i i y du ing de elopmen , s ains con aining
lacZ usions (Figu e 2) we e cul u ed and spo ed on o CF pla es as desc ibed abo e. Cell ex ac s
we e ob ained a di e en imes by sonica ion and assayed o ac i i y as p e iously epo ed
(Mo aleda-Mun
˜oz e al., 2003). The amoun o p o ein in he supe na an s was de e mined by using
he Bio-Rad P o ein Assay (Bio-Rad, Inc) wi h bo ine se um albumin as a s anda d. Speci ic ac i i y is
exp essed as nmol o o-ni ophenol (ONP) p oduced pe min and mg o p o ein. The esul s a e he
a e age and associa ed s anda d de ia ion om h ee independen biological eplica es.
Mic oscopy
To obse e swa m and ui ing bodies, 10 ml o cell suspension p epa ed as men ioned abo e we e
spo ed on o CTT ( o g ow h) o CF ( o de elopmen ) aga pla es and incuba ed a 30˚C. Obse a-
ion was on an Olympus SZX7 dissec ing mic oscope. Fo scanning elec on mic oscopy, samples
ob ained om CF and CTT aga pla es we e ixed wi h glu a aldehyde apo s o 24 h a oom em-
pe a u e and hen pos ixed in aqueous 1% osmium e oxide o 1 h a 4˚C, washed h ee imes in
bu e , and pos s ained o 2 h in bu e ed 0.5% u anyl ace a e. Dehyd a ion was accomplished by a
g aded se ies o e hanol. Samples we e hen c i ical-poin d ied and spu e coa ed wi h gold. Pho-
og aphs we e aken in a Zeiss DSM950 scanning elec on mic oscope.
Suppo ing da a
The ansc ip ome sequencing da a ( aw- eads) was submi ed o NCBI SRA unde he Biop ojec
accession numbe : PRJNA493545. SRA accession numbe s o each o he eplicas a e as ollows: 0
h : SAMN10135973 (WT_0_1-biological_ eplica e_1) and SAMN10135974 (WT_0_2-biological_ epli-
ca e_2); 6 h : SAMN10135975 (WT_6_1-biological_ eplica e_1) and SAMN10135976 (WT_6_2-bio-
logical_ eplica e_2); 12 h : SAMN10135977 (WT_12_1-biological_ eplica e_1) and SAMN10135978
(WT_12_2-biological_ eplica e_2); 24 h : SAMN10135979 (WT_24_1-biological_ eplica e_1) and
SAMN10135980 (WT_24_2-biological_ eplica e_2); 48 h : SAMN10135981 (WT_48_1-biological_ e-
plica e_1) and SAMN10135982 (WT_48_2-biological_ eplica e_2); 72 h : SAMN10135983 (WT_72_1-
biological_ eplica e_1) and SAMN10135984 (WT_72_2-biological_ eplica e_2); 96 h :
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Tools and esou ces Mic obiology and In ec ious Disease

SAMN10135985 (WT_96_1-biological_ eplica e_1) and SAMN10135986 (WT_96_2-
biological_ eplica e_2).
Acknowledgemen s
This wo k has been suppo ed by he Spanish Go e nmen (g an s CSD2009-00006 o JMD and
BFU2016-75425-P o AMM [70% unded by FEDER]), and by NIGMS o he Na ional Ins i u es o
Heal h unde awa d numbe R35GM124733 o JMS. JPT and JMD we e g an ed wi h ellowships o
he Sal ado de Mada iaga P og am o s ay a Wayne S a e Uni e si y o ou mon hs. PIH was
unded by a g an om NSF IOS 1651921. We also wan o hank P o . Lee K oos o p o iding
s ains.
Addi ional in o ma ion
Funding
Funde G an e e ence numbe Au ho
Spanish Go e nmen CSD2009-00006 Jose Munoz-Do ado
Spanish Go e nmen BFU2016-75425-P Au elio Mo aleda-Mun
˜oz
Na ional Ins i u es o Heal h R35GM124733 Ja ed M Sch ade
Na ional Science Founda ion IOS 1651921 Penelope I Higgs
Minis e io de Educacio
´n, Cul-
u a y Depo e
Sal ado de Mada iaga
P og am
Jose Munoz-Do ado
Juana Pe
´ ez
The unde s had no ole in s udy design, da a collec ion and in e p e a ion, o he
decision o submi he wo k o publica ion.
Au ho con ibu ions
Jose´ Mun
˜oz-Do ado, Juana Pe´ ez, Subs an ial con ibu ions o concep ion and design, acquisi ion o
da a, and analysis and in e p e a ion o da a; D a ing he a icle and e ising i c i ically o
impo an in ellec ual con en ; Final app o al o he e sion o be published; Au elio Mo aleda-
Mun
˜oz, F ancisco Ja ie Ma cos-To es, F ancisco Ja ie Con e as-Mo eno, Acquisi ion o da a;
Re ising he a icle c i ically o impo an in ellec ual con en ; Final app o al o he e sion o be
published; Ana Belen Ma in-Cuad ado, Ja ed M Sch ade , Acquisi ion o da a and analysis and
in e p e a ion o da a; Re ising he a icle c i ically o impo an in ellec ual con en ; Final app o al
o he e sion o be published; Penelope I Higgs, Analysis and in e p e a ion o da a; D a ing he
a icle and e ising i c i ically o impo an in ellec ual con en ; Final app o al o he e sion o be
published
Au ho ORCIDs
Jose´ Mun
˜oz-Do ado h ps://o cid.o g/0000-0001-7765-5687
Ja ed M Sch ade h ps://o cid.o g/0000-0002-5728-5882
Decision le e and Au ho esponse
Decision le e h ps://doi.o g/10.7554/eLi e.50374.033
Au ho esponse h ps://doi.o g/10.7554/eLi e.50374.034
Addi ional iles
Supplemen a y iles
.T anspa en epo ing o m
DOI: h ps://doi.o g/10.7554/eLi e.50374.029
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Tools and esou ces Mic obiology and In ec ious Disease
Da a a ailabili y
The ansc ip ome sequencing da a ( aw- eads) was submi ed o NCBI SRA unde he Biop ojec
accession numbe : PRJNA493545. SRA accession numbe s o each o he eplicas a e as ollows: 0
h: SAMN10135973 (WT_0_1-biological_ eplica e_1) and SAMN10135974 (WT_0_2-biological_ epli-
ca e_2); 6 h: SAMN10135975 (WT_6_1-biological_ eplica e_1) and SAMN10135976 (WT_6_2-biolog-
ical_ eplica e_2); 12 h: SAMN10135977 (WT_12_1-biological_ eplica e_1) and SAMN10135978
(WT_12_2-biological_ eplica e_2); 24 h: SAMN10135979 (WT_24_1-biological_ eplica e_1) and
SAMN10135980 (WT_24_2-biological_ eplica e_2); 48 h: SAMN10135981 (WT_48_1-biological_ epli-
ca e_1) and SAMN10135982 (WT_48_2-biological_ eplica e_2); 72 h: SAMN10135983 (WT_72_1-bio-
logical_ eplica e_1) and SAMN10135984 (WT_72_2-biological_ eplica e_2); 96 h: SAMN10135985
(WT_96_1-biological_ eplica e_1) and SAMN10135986 (WT_96_2-biological_ eplica e_2).
The ollowing da ase was gene a ed:
Au ho (s) Yea Da ase i le Da ase URL
Da abase and
Iden i ie
Ma in-Cuad ado,
Ana-Belen
2018 Myxococcus xan hus DK1622
ansc ip ome (TaxID: 34)
h ps://www.ncbi.nlm.
nih.go /biop ojec /
PRJNA493545
NCBI BioP ojec ,
PRJNA493545
Re e ences
Apelian D, Inouye S. 1993. A new pu a i e sigma ac o o Myxococcus xan hus.Jou nal o Bac e iology 175:
3335–3342. DOI: h ps://doi.o g/10.1128/jb.175.11.3335-3342.1993,PMID: 8501037
Bha S, Boyn on TO, Pham D, Shimke s LJ. 2014. Fa y acids om memb ane lipids become inco po a ed in o
lipid bodies du ing Myxococcus xan hus di e en ia ion. PLOS ONE 9:e99622. DOI: h ps://doi.o g/10.1371/
jou nal.pone.0099622,PMID: 24906161
Bode HB, Ring MW, Schwa
¨ G, Al meye MO, Kegle C, Jose IR, Singe M, Mu
¨lle R. 2009. Iden i ica ion o
addi ional playe s in he al e na i e biosyn hesis pa hway o iso ale yl-CoA in he myxobac e ium Myxococcus
xan hus.ChemBioChem 10:128–140. DOI: h ps://doi.o g/10.1002/cbic.200800219,PMID: 18846531
B e l DJ, Ki by JR. 2016. Molecula mechanisms o signaling in Myxococcus xan hus De elopmen . Jou nal o
Molecula Biology 428:3805–3830. DOI: h ps://doi.o g/10.1016/j.jmb.2016.07.008,PMID: 27430596
B e sche AP, Kaise D. 1978. Nu i ion o Myxococcus xan hus, a ui ing myxobac e ium. Jou nal o
Bac e iology 133:763–768. PMID: 415048
Campoy S, Fon es M, Padmanabhan S, Co e´ s P, Llagos e a M, Ba be´ J. 2003. LexA-independen DNA damage-
media ed induc ion o gene exp ession in Myxococcus xan hus.Molecula Mic obiology 49:769–781.
DOI: h ps://doi.o g/10.1046/j.1365-2958.2003.03592.x,PMID: 12864858
Chang YW, Re be g LA, T eune -Lange A, Iwasa J, Søgaa d-Ande sen L, Jensen GJ. 2016. A chi ec u e o he
ype IVa pilus machine. Science 351:aad2001. DOI: h ps://doi.o g/10.1126/science.aad2001,PMID: 26965631
Cha i a M, Cao N, Le K, Ria T, Mo adshahi N, McB ide M, Lux R, Shi W. 2007. Be a-D-Allose inhibi s ui ing
body o ma ion and spo ula ion in Myxococcus xan hus.Jou nal o Bac e iology 189:169–178. DOI: h ps://doi.
o g/10.1128/JB.00792-06,PMID: 17056749
Cu is PD, Shimke s LJ. 2008. Me abolic pa hways ele an o p eda ion, signaling, and de elopmen . In:
Whi wo h D. E (Ed). Myxobac e ia: Mul icellula i y and Di e en ia ion. ASM P ess.
de Hoon MJ, Imo o S, Nolan J, Miyano S. 2004. Open sou ce clus e ing so wa e. Bioin o ma ics 20:1453–1454.
DOI: h ps://doi.o g/10.1093/bioin o ma ics/b h078,PMID: 14871861
Dioda i ME, Gill RE, Plamann L, Singe M. 2008. Ini ia ion and ea ly de elopmen al e en s. In: Whi wo h D. E
(Ed). Myxobac e ia: Mul icellula i y and Di e en ia ion. ASM P ess.
Dwo kin M, Gibson SM. 1964. A sys em o s udying mic obial mo phogenesis: apid o ma ion o mic ocys s in
Myxococcus xan hus.Science 146:243–244. DOI: h ps://doi.o g/10.1126/science.146.3641.243,PMID: 141
85314
Fawce P, Eichenbe ge P, Losick R, Youngman P. 2000. The ansc ip ional p o ile o ea ly o middle
spo ula ion in Bacillus sub ilis.PNAS 97:8063–8068. DOI: h ps://doi.o g/10.1073/pnas.140209597,PMID: 1086
9437
Fos e HA, Pa ish JH. 1973. Ribosomes, ibosomal subuni s and ibosomal p o eins om Myxococcus xan hus.
Jou nal o Gene al Mic obiology 75:391–400. DOI: h ps://doi.o g/10.1099/00221287-75-2-391
Fu G, Banda ia JN, Le Gall AV, Fan X, Yildiz A, Migno T, Zusman DR, Nan B. 2018. Mo AB-like machine y d i es
he mo emen o M eB ilamen s du ing bac e ial gliding mo ili y. PNAS 115:2484–2489. DOI: h ps://doi.o g/
10.1073/pnas.1716441115
Ge sin I, Nalbandian GH, Yee DC, Vas e ma k A, Papa odi is PC, Reddy VS, Saie MH. 2013. Compa a i e
genomics o anspo p o eins in de elopmen al Bac e ia: Myxococcus xan hus and S ep omyces coelicolo .
BMC Mic obiology 13:279. DOI: h ps://doi.o g/10.1186/1471-2180-13-279,PMID: 24304716
Mun
˜oz-Do ado e al. eLi e 2019;8:e50374. DOI: h ps://doi.o g/10.7554/eLi e.50374 23 o 27
Tools and esou ces Mic obiology and In ec ious Disease
Giglio KM, Cabe oy N, Suen G, Kaise D, Ga za AG. 2011. A cascade o co egula ing enhance binding p o eins
ini ia es and p opaga es a mul icellula de elopmen al p og am. PNAS 108:E431–E439. DOI: h ps://doi.o g/
10.1073/pnas.1105876108,PMID: 21670274
Goha a DW, Yap MF. 2018. Su i al o he d owsies : he hibe na ing 100S ibosome in bac e ial s ess
managemen . Cu en Gene ics 64:753–760. DOI: h ps://doi.o g/10.1007/s00294-017-0796-2,PMID: 2
9243175
Goldman BS, Nie man WC, Kaise D, Sla e SC, Du kin AS, Eisen JA, Eisen J, Ronning CM, Ba bazuk WB,
Blancha d M, Field C, Halling C, Hinkle G, Ia chuk O, Kim HS, Mackenzie C, Madupu R, Mille N, Sh a sbeyn
A, Sulli an SA, e al. 2006. E olu ion o senso y complexi y eco ded in a myxobac e ial genome. PNAS 103:
15200–15205. DOI: h ps://doi.o g/10.1073/pnas.0607335103,PMID: 17015832
Hagen DC, B e sche AP, Kaise D. 1978. Syne gism be ween mo phogene ic mu an s o Myxococcus xan hus.
De elopmen al Biology 64:284–296. DOI: h ps://doi.o g/10.1016/0012-1606(78)90079-9,PMID: 98366
Ha ms A, Maisonneu e E, Ge des K. 2016. Mechanisms o bac e ial pe sis ence du ing s ess and an ibio ic
exposu e. Science 354:aa 4268. DOI: h ps://doi.o g/10.1126/science.aa 4268,PMID: 27980159
Hensley MP, Gunaseke a TS, Eas on JA, Sigdel TK, Suga bake SA, Klingbeil L, B eece RM, Tie ney DL, C owde
MW. 2012. Cha ac e iza ion o zn(II)- esponsi e ibosomal p o eins YkgM and L31 in E. coli.Jou nal o
Ino ganic Biochemis y 111:164–172. DOI: h ps://doi.o g/10.1016/j.jino gbio.2011.11.022,PMID: 22196016
Higgs PI, Ha zell P, Holkenb ink C, Hoiczyk E. 2014. Myxococcus xan hus ege a i e and de elopmen al cell
he e ogenei y. In: Yang Z, Higgs P. I (Eds). Myxobac e ia: Genomics, Cellula and Molecula Biology. Cais e
Academic P ess.
Hodgkin J, Kaise D. 1977. Cell- o-cell s imula ion o mo emen in nonmo ile mu an s o Myxococcus.PNAS 74:
2938–2942. DOI: h ps://doi.o g/10.1073/pnas.74.7.2938,PMID: 16592422
Hoiczyk E, Ring MW, McHugh CA, Schwa
¨ G, Bode E, K ug D, Al meye MO, Lu JZ, Bode HB. 2009. Lipid body
o ma ion plays a cen al ole in cell a e de e mina ion du ing de elopmen al di e en ia ion o Myxococcus
xan hus.Molecula Mic obiology 74:497–517. DOI: h ps://doi.o g/10.1111/j.1365-2958.2009.06879.x,PMID: 1
9788540
Holkenb ink C, Hoiczyk E, Kahn J, Higgs PI. 2014. Syn hesis and assembly o a no el glycan laye in Myxococcus
xan hus spo es. The Jou nal o Biological Chemis y 289:32364–32378. DOI: h ps://doi.o g/10.1074/jbc.M114.
595504,PMID: 25271164
Inouye S. 1990. Cloning and DNA sequence o he gene coding o he majo sigma ac o om Myxococcus
xan hus.Jou nal o Bac e iology 172:80–85. DOI: h ps://doi.o g/10.1128/jb.172.1.80-85.1990,PMID: 2104614
Inouye S, Na iya H, Mun
˜oz-Do ado J. 2008. P o ein Se /Th kinases and phospha ases in Myxococcus xan hus. In:
Whi wo h D. E (Ed). Myxobac e ia: Mu icellula i y and Di e en ia ion. ASM P ess.
Islam ST, Migno T. 2015. The mys e ious na u e o bac e ial su ace (gliding) mo ili y: a ocal adhesion-based
mechanism in Myxococcus xan hus.Semina s in Cell & De elopmen al Biology 46:143–154. DOI: h ps://doi.
o g/10.1016/j.semcdb.2015.10.033,PMID: 26520023
Jakobsen JS, Jelsbak L, Jelsbak L, Welch RD, Cummings C, Goldman B, S a k E, Sla e S, Kaise D. 2004.
Sigma54 enhance binding p o eins and Myxococcus xan hus ui ing body de elopmen . Jou nal o
Bac e iology 186:4361–4368. DOI: h ps://doi.o g/10.1128/JB.186.13.4361-4368.2004,PMID: 15205438
Kaise D. 1979. Social gliding is co ela ed wi h he p esence o pili in Myxococcus xan hus.PNAS 76:5952–
5956. DOI: h ps://doi.o g/10.1073/pnas.76.11.5952,PMID: 42906
Kesele IM, Kaise D. 1997. sigma54, a i al p o ein o Myxococcus xan hus.PNAS 94:1979–1984. DOI: h ps://
doi.o g/10.1073/pnas.94.5.1979,PMID: 9050890
Kono alo a A, Wegene -Feldb u
¨gge S, Søgaa d-Ande sen L. 2012. Two in e cellula signals equi ed o ui ing
body o ma ion in Myxococcus xan hus ac sequen ially bu non-hie a chically. Molecula Mic obiology 86:65–
81. DOI: h ps://doi.o g/10.1111/j.1365-2958.2012.08173.x,PMID: 22834948
Ko p J, Vela Gu o ic MS, Ne M. 2016. An ibio ics om p eda o y bac e ia. Beils ein Jou nal o O ganic
Chemis y 12:594–607. DOI: h ps://doi.o g/10.3762/bjoc.12.58,PMID: 27340451
Ko el RH, Bacon K, Clu e D, Whi e D. 1975. Coa s om Myxococcus xan hus: cha ac e iza ion and syn hesis
du ing myxospo e di e en ia ion. Jou nal o Bac e iology 124:550–557. PMID: 809426
K oos L, Kuspa A, Kaise D. 1986. A global analysis o de elopmen ally egula ed genes in Myxococcus xan hus.
De elopmen al Biology 117:252–266. DOI: h ps://doi.o g/10.1016/0012-1606(86)90368-4,PMID: 3017794
K oos L. 2017. Highly Signal-Responsi e gene egula o y ne wo k go e ning Myxococcus De elopmen . T ends
in Gene ics 33:3–15. DOI: h ps://doi.o g/10.1016/j. ig.2016.10.006,PMID: 27916428
Laub MT, McAdams HH, Feldblyum T, F ase CM, Shapi o L. 2000. Global analysis o he gene ic ne wo k
con olling a bac e ial cell cycle. Science 290:2144–2148. DOI: h ps://doi.o g/10.1126/science.290.5499.2144,
PMID: 11118148
Leona dy S, F eyma k G, Hebene S, Ellehauge E, Søgaa d-Ande sen L. 2007. Coupling o p o ein localiza ion
and cell mo emen s by a dynamically localized esponse egula o in Myxococcus xan hus.The EMBO Jou nal
26:4433–4444. DOI: h ps://doi.o g/10.1038/sj.emboj.7601877,PMID: 17932488
Li Y, Sun H, Ma X, Lu A, Lux R, Zusman D, Shi W. 2003. Ex acellula polysaccha ides media e Pilus e ac ion
du ing social mo ili y o Myxococcus xan hus.PNAS 100:5443–5448. DOI: h ps://doi.o g/10.1073/pnas.
0836639100,PMID: 12704238
Li H, Handsake B, Wysoke A, Fennell T, Ruan J, Home N, Ma h G, Abecasis G, Du bin R, 1000 Genome
P ojec Da a P ocessing Subg oup. 2009. The sequence alignmen /Map o ma and SAM ools. Bioin o ma ics
25:2078–2079. DOI: h ps://doi.o g/10.1093/bioin o ma ics/b p352,PMID: 19505943
Mun
˜oz-Do ado e al. eLi e 2019;8:e50374. DOI: h ps://doi.o g/10.7554/eLi e.50374 24 o 27
Tools and esou ces Mic obiology and In ec ious Disease
Li H, Du bin R. 2009. Fas and accu a e sho ead alignmen wi h Bu ows-Wheele ans o m. Bioin o ma ics 25:
1754–1760. DOI: h ps://doi.o g/10.1093/bioin o ma ics/b p324,PMID: 19451168
Lobedanz S, Søgaa d-Ande sen L. 2003. Iden i ica ion o he C-signal, a con ac -dependen mo phogen
coo dina ing mul iple de elopmen al esponses in Myxococcus xan hus.Genes & De elopmen 17:2151–2161.
DOI: h ps://doi.o g/10.1101/gad.274203,PMID: 12923062
Lux R, Shi W. 2005. A no el bac e ial signalling sys em wi h a combina ion o a se /Th kinase cascade and a his/
Asp wo-componen sys em. Molecula Mic obiology 58:345–348. DOI: h ps://doi.o g/10.1111/j.1365-2958.
2005.04856.x,PMID: 16194223
Mau iello EM, Migno T, Yang Z, Zusman DR. 2010. Gliding mo ili y e isi ed: how do he myxobac e ia mo e
wi hou lagella? Mic obiology and Molecula Biology Re iews 74:229–249. DOI: h ps://doi.o g/10.1128/
MMBR.00043-09,PMID: 20508248
McB ide MJ, Zusman DR. 1989. T ehalose accumula ion in ege a i e cells and spo es o Myxococcus xan hus.
Jou nal o Bac e iology 171:6383–6386. DOI: h ps://doi.o g/10.1128/jb.171.11.6383-6386.1989,PMID: 250
9436
McLaughlin PT, Bha dwaj V, Feeley BE, Higgs PI. 2018. M pC, a CRP/Fn homolog, unc ions as a nega i e
au o egula o du ing he Myxococcus xan hus mul icellula de elopmen al p og am . Molecula Mic obiology
109:245–261. DOI: h ps://doi.o g/10.1111/mmi.13982
Me cie R, Migno T. 2016. Regula ions go e ning he mul icellula li es yle o Myxococcus xan hus.Cu en
Opinion in Mic obiology 34:104–110. DOI: h ps://doi.o g/10.1016/j.mib.2016.08.009,PMID: 27648756
Mo aleda-Mun
˜oz A, Ca e o-Le´ ida J, Pe´ ez J, Mun
˜oz-Do ado J. 2003. Role o wo no el wo-componen
egula o y sys ems in de elopmen and phospha ase exp ession in Myxococcus xan hus.Jou nal o
Bac e iology 185:1376–1383. DOI: h ps://doi.o g/10.1128/JB.185.4.1376-1383.2003,PMID: 12562808
Mo e E, Sego ia L. 1993. The sigma 54 bac e ial enhance -binding p o ein amily: mechanism o ac ion and
phylogene ic ela ionship o hei unc ional domains. Jou nal o Bac e iology 175:6067–6074. DOI: h ps://doi.
o g/10.1128/jb.175.19.6067-6074.1993,PMID: 8407777
Mu
¨lle FD, T eune -Lange A, Heide J, Hun ley SM, Higgs PI. 2010. Global ansc ip ome analysis o spo e
o ma ion in Myxococcus xan hus e eals a locus necessa y o cell di e en ia ion. BMC Genomics 11:264.
DOI: h ps://doi.o g/10.1186/1471-2164-11-264,PMID: 20420673
Mu
¨lle FD, Schink CW, Hoiczyk E, Cse i E, Higgs PI. 2012. Spo e o ma ion in Myxococcus xan hus is ied o
cy oskele on unc ions and polysaccha ide spo e coa deposi ion. Molecula Mic obiology 83:486–505.
DOI: h ps://doi.o g/10.1111/j.1365-2958.2011.07944.x,PMID: 22188356
Mu
¨lle S, S ack SN, Ryan SE, Shawgo M, Walling A, Ha is S, Chambe s C, Boddicke J, Ki by JR. 2016.
Iden i ica ion o unc ions a ec ing P eda o -P ey in e ac ions be ween Myxococcus xan hus and Bacillus
sub ilis.Jou nal o Bac e iology 198:3335–3344. DOI: h ps://doi.o g/10.1128/JB.00575-16,PMID: 27698086
Mun
˜oz-Do ado J, Inouye S, Inouye M. 1991. A gene encoding a p o ein se ine/ h eonine kinase is equi ed o
no mal de elopmen o M. xan hus, a g am-nega i e bac e ium. Cell 67:995–1006. DOI: h ps://doi.o g/10.
1016/0092-8674(91)90372-6,PMID: 1835671
Mun
˜oz-Do ado J, Higgs PI, Elı
´as-A nanz M. 2014. Abundance and complexi y o signaling mechanisms in
myxobac e ia. In: Yang Z, Higgs P. I (Eds). Myxobac e ia: Genomics Cellula and Molecula Biology. Cais e
Academic P ess.
Mun
˜oz-Do ado J, Ma cos-To es FJ, Ga cı
´a-B a o E, Mo aleda-Mun
˜oz A, Pe´ ez J. 2016. Myxobac e ia: mo ing,
killing, eeding, and su i ing oge he . F on ie s in Mic obiology 7:781. DOI: h ps://doi.o g/10.3389/ micb.
2016.00781,PMID: 27303375
Nan B, McB ide MJ, Chen J, Zusman DR, Os e G. 2014. Bac e ia ha glide wi h helical acks. Cu en Biology
24:R169–R173. DOI: h ps://doi.o g/10.1016/j.cub.2013.12.034,PMID: 24556443
Nanamiya H, Akanuma G, Na o i Y, Mu ayama R, Kosono S, Kudo T, Kobayashi K, Ogasawa a N, Pa k SM, Ochi
K, Kawamu a F. 2004. Zinc is a key ac o in con olling al e na ion o wo ypes o L31 p o ein in he Bacillus
sub ilis ibosome. Molecula Mic obiology 52:273–283. DOI: h ps://doi.o g/10.1111/j.1365-2958.2003.03972.x,
PMID: 15049826
Na iya H, Inouye S. 2003. An e ec i e spo ula ion o Myxococcus xan hus equi es glycogen consump ion ia
Pkn4-ac i a ed 6-phospho uc okinase. Molecula Mic obiology 49:517–528. DOI: h ps://doi.o g/10.1046/j.
1365-2958.2003.03572.x,PMID: 12828646
Na iya H, Inouye M. 2008. MazF, an mRNA in e e ase, media es p og ammed cell dea h du ing mul icellula
Myxococcus de elopmen . Cell 132:55–66. DOI: h ps://doi.o g/10.1016/j.cell.2007.11.044,PMID: 18191220
O’Conno KA, Zusman DR. 1991a. Analysis o Myxococcus xan hus cell ypes by wo-dimensional polyac ylamide
gel elec opho esis. Jou nal o Bac e iology 173:3334–3341. DOI: h ps://doi.o g/10.1128/jb.173.11.3334-
3341.1991,PMID: 1904431
O’Conno KA, Zusman DR. 1991b. Beha io o pe iphe al ods and hei ole in he li e cycle o Myxococcus
xan hus.Jou nal o Bac e iology 173:3342–3355. DOI: h ps://doi.o g/10.1128/jb.173.11.3342-3355.1991,
PMID: 1904432
O’Conno KA, Zusman DR. 1991c. De elopmen in Myxococcus xan hus in ol es di e en ia ion in o wo cell
ypes, pe iphe al ods and spo es. Jou nal o Bac e iology 173:3318–3333. DOI: h ps://doi.o g/10.1128/jb.
173.11.3318-3333.1991,PMID: 1904430
Pe
´ ez J, Cas an
˜eda-Ga cı
´a A, Jenke-Kodama H, Mu
¨lle R, Mun
˜oz-Do ado J. 2008. Euka yo ic-like p o ein kinases
in he p oka yo es and he myxobac e ial kinome. PNAS 105:15950–15955. DOI: h ps://doi.o g/10.1073/pnas.
0806851105,PMID: 18836084
Mun
˜oz-Do ado e al. eLi e 2019;8:e50374. DOI: h ps://doi.o g/10.7554/eLi e.50374 25 o 27
Tools and esou ces Mic obiology and In ec ious Disease