micb-14-1213659 June 13, 2023 Time: 16:41 # 1
TYPE O iginal Resea ch
PUBLISHED 19 June 2023
DOI 10.3389/ micb.2023.1213659
OPEN ACCESS
EDITED BY
Ch is ian Sohlenkamp,
Na ional Au onomous Uni e si y o Mexico,
Mexico
REVIEWED BY
Da id Cole S e ens,
Uni e si y o Mississippi, Uni ed S a es
Alessio Mengoni,
Uni e si y o Flo ence, I aly
Ch is ine Kaime ,
Ruh Uni e si y Bochum, Ge many
*CORRESPONDENCE
Au elio Mo aleda-Muñoz
au eliom@ug .es
†These au ho s ha e con ibu ed equally o his
wo k
RECEIVED 28 Ap il 2023
ACCEPTED 01 June 2023
PUBLISHED 19 June 2023
CITATION
So o MJ, Pé ez J, Muñoz-Do ado J,
Con e as-Mo eno FJ and Mo aleda-Muñoz A
(2023) T ansc ip omic esponse
o Sino hizobium melilo i o he p eda o y
a ack o Myxococcus xan hus.
F on . Mic obiol. 14:1213659.
doi: 10.3389/ micb.2023.1213659
COPYRIGHT
© 2023 So o, Pé ez, Muñoz-Do ado,
Con e as-Mo eno and Mo aleda-Muñoz. This
is an open-access a icle dis ibu ed unde he
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License (CC BY). The use, dis ibu ion o
ep oduc ion in o he o ums is pe mi ed,
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acco dance wi h accep ed academic p ac ice.
No use, dis ibu ion o ep oduc ion is
pe mi ed which does no comply wi h
hese e ms.
T ansc ip omic esponse o
Sino hizobium melilo i o he
p eda o y a ack o Myxococcus
xan hus
Ma ía José So o1†, Juana Pé ez2†, José Muñoz-Do ado2,
F ancisco Ja ie Con e as-Mo eno2and
Au elio Mo aleda-Muñoz2*
1Depa amen o de Bio ecnología y P o ección Ambien al, Es ación Expe imen al del Zaidín, Consejo
Supe io de In es igaciones Cien í icas, G anada, Spain, 2Depa amen o de Mic obiología, Facul ad
de Ciencias, Uni e sidad de G anada, G anada, Spain
Bac e ial p eda ion impac s mic obial communi y s uc u es, which can ha e bo h
posi i e and nega i e e ec s on plan and animal heal h and on en i onmen al
sus ainabili y. Myxococcus xan hus is an epibio ic soil p eda o wi h a b oad
ange o p ey, including Sino hizobium melilo i, which es ablishes ni ogen-
ixing symbiosis wi h legumes. Du ing he M. xan hus-S. melilo i in e ac ion, he
p eda o mus adap i s ansc ip ome o kill and lyse he a ge (p eda osome),
and he p ey mus o ches a e a ansc ip ional esponse (de ensome) o p o ec
i sel agains he bio ic s ess caused by he p eda o y a ack. He e, we desc ibe
he ansc ip ional changes aking place in S. melilo i in esponse o myxobac e ial
p eda ion. The esul s indica e ha he p eda o induces massi e changes in
he p ey ansc ip ome wi h up- egula ion o p o ein syn hesis and sec e ion,
ene gy gene a ion, and a y acid (FA) syn hesis, while down- egula ing genes
equi ed o FA deg ada ion and ca bohyd a e anspo and me abolism. The
econs uc ion o up- egula ed pa hways sugges s ha S. melilo i modi ies he
cell en elop by inc easing he p oduc ion o di e en su ace polysaccha ides
(SPSs) and memb ane lipids. Besides he ba ie ole o SPSs, addi ional
mechanisms in ol ing he ac i i y o e lux pumps and he pep ide up ake
anspo e BacA, oge he wi h he p oduc ion o H2O2and o maldehyde ha e
been un eiled. Also, he induc ion o he i on-up ake machine y in bo h p eda o
and p ey e lec s a s ong compe i ion o his me al. Wi h his esea ch we
comple e he cha ac e iza ion o he complex ansc ip ional changes ha occu
du ing he M. xan hus-S. melilo i in e ac ion, which can impac he es ablishmen
o bene icial symbiosis wi h legumes.
KEYWORDS
bac e ial p eda ion, myxobac e ia, Sino hizobium melilo i, de ensome, bac e ial
in e ac ions
1. In oduc ion
Bac e ia in e ac wi h co-habi ing mic obes in di e en mul ispecies communi ies. The
ecological and e olu iona y success o mic oo ganisms in a pa icula en i onmen is no
only go e ned by hei capaci y o adap o ex e nal abio ic s esses, bu also depends on
hei abili y o de ec and espond o compe i ion wi h he neighbo ing cells. Consequen ly,
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So o e al. 10.3389/ micb.2023.1213659
he me abolic p ocesses o one s ain a e in luenced by he
me abolic unc ions o he o he membe s o he communi y.
The ela ionships be ween mic oo ganisms ange om coope a i e
symbio ic associa ions o di e en compe i ion s a egies. In all
cases, bac e ial in e ac ions in ol e complex p ocesses ha a e
key de e minan s ha s ongly shape he s uc u e o bac e ial
communi ies (Baue e al., 2018;G ana o e al., 2019). In he
las decades many s udies ha e e ealed how bac e ial in e ac ions
ha occu in small communi ies ha e consequences ha a ec in
many cases human, animal, and plan heal h (Pé ez e al., 2011;
S ubbendieck e al., 2016;Niehaus e al., 2019;Molina-San iago
e al., 2021;Ma ins e al., 2022).
A pa icula ype o in e ac ion is ep esen ed by p eda o y
bac e ia, which a e species ha kill and lyse suscep ible cells in
o de o consume he cellula ma e ials as ca bon and ene gy
sou ces (Pé ez e al., 2016;Whi wo h e al., 2020). Mos bac e ial
p eda o s use wo majo app oaches o kill p ey: (i) he endobio ic
s a egy ep esen ed by Bdello ib io and like o ganisms (BALOs)
ha mainly p ey on dide m bac e ia in he plank onic phase as
well as in bio ilms (Mookhe jee and Ju ke i ch, 2022), and (ii) he
epibio ic p eda ion exempli ied by myxobac e ia ha can kill and
ex e nally lyse a g ea a ie y o mic oo ganisms (Muñoz-Do ado
e al., 2016;Pé ez e al., 2016).
The capaci y o bac e ial p eda o s o kill o he bac e ia,
including mul id ug- esis an pa hogens, has a ac ed he
a en ion o esea che s as a easible al e na i e o an ibio ics in
he ac ual c isis (Pé ez e al., 2020). As he use o echnologies
such as nex gene a ion sequencing and me a- ansc ip omics a e
inc easingly being applied o he s udy o soil mic obio a, bac e ial
p eda ion is gaining ele an impo ance as a shape o mic obial
communi ies. Al hough myxobac e ia ha e been adi ionally
conside ed mino i y componen s o soil bac e ial communi ies,
se e al s udies ha e e ealed ha his does no seem o be he
case, and ha he myxobac e ial communi y is a p edominan and
highly di e se g oup wi hin soil niches (Zhou e al., 2014). Un il
ecen ly, p o is s ha e been conside ed he dominan g oup p eying
on bac e ia. Howe e , he esul s o ecen s udies s ongly sugges
he impo ance, and possibly e en dominance, o myxobac e ia
as soil p eda o s. In ac , an analysis o 28 Eu opean soils showed
ha in mos o hese soils myxobac e ia comp ise 1.5–9.7% o all
ob ained SSU RNA ansc ip s and mo e han 60% o all iden i ied
po en ial bac e i o es (Pe e s e al., 2021).
Myxococcus xan hus is a soil myxobac e ium which has been
ex ensi ely s udied because o i s unique complex li ecycle. This
li ecycle consis s o wo s ages: a ege a i e g ow h s age in he
p esence o nu ien s and/o p ey (i is a acul a i e p eda o ); and
a de elopmen al s age (wi h he o ma ion o mac oscopic ui ing
bodies illed o myxospo es) when nu ien s a e deple ed (Muñoz-
Do ado e al., 2016). I has a la ge genome which encodes all
he genes ha pa icipa e in he complex social and mul icellula
li es yle exhibi ed du ing bo h g ow h and de elopmen (Goldman
e al., 2006).
Myxococcus xan hus p eda ion equi es he pa icipa ion o
many weapons o kill and consume he p ey, including a a ie y o
hyd oly ic enzymes, ou e memb ane esicles, con ac -dependen
and independen elemen s, and he p oduc ion o seconda y
me aboli es such as an ibio ics (Pé ez e al., 2016;Thie y and
Kaime , 2020;See e al., 2021). In addi ion, his a senal di e s om
one p ey o ano he (Thie y e al., 2022).
In ou labo a o y we deciphe ed he ansc ip omic changes
ha ake place in M. xan hus du ing a comple e li ecycle. Du ing
de elopmen , 1,415 genes we e sequen ially and di e en ially
exp essed in 10 disc e e g oups (Muñoz-Do ado e al., 2019).
Mo eo e , we ha e also analyzed he p eda osome o his
myxobac e ium when p eying on Sino hizobium melilo i. The
esul s ob ained e ealed ha he numbe o genes di e en ially
exp essed du ing p eda ion is lowe han du ing de elopmen .
Among he ansc ip s ha a e up- egula ed du ing p eda ion,
he mos no ewo hy a e genes in ol ed in he biosyn hesis
o seconda y me aboli es, in he syn hesis and deg ada ion o
lipids, hose encoding bo h ex acellula and ou e memb ane
hyd oly ic enzymes, and genes ela ed o social mo ili y and Tad-
like appa a uses (Pé ez e al., 2022).
In p eda o y in e ac ions, he p ey will need o modi y he
ne wo k o genes and pa hways equi ed o moun an o ches a ed
de ense agains he bio ic s ess caused by he p eda o y a ack.
We will use he e m “de ensome” o e e o he whole se o
genes ha a y hei ansc ip ion in esponse o he p eda o y
bac e ium, ei he o esis p eda ion o o compe e o esou ces.
Ne e heless, some speci ic de ense mechanisms and adap a ions o
di e en p ey agains M. xan hus a ack ha e been epo ed, so a .
Fo example, galac oglucan and melanin p o ec S. melilo i om
p eda ion by his myxobac e ium (Pé ez e al., 2014;Con e as-
Mo eno e al., 2020). The p esence o his p eda o also induces he
ansc ip ional ac i a ion o silenced genes coding o an ibio ics
in S ep omyces coelicolo (Pé ez e al., 2011;Lee e al., 2020).
Bacillus sub ilis induces bacillaene syn hesis and o ms spo e- illed
megas uc u es agains he a ack o M. xan hus (Mülle e al.,
2014, 2015). On he o he hand, s udies o he p ey esponse
e ealed a no el an ibio ic esis ance mechanism consis ing o
he glucosyla ion o he an ibio ic myxo i escin TA, which was
disco e ed in Bacillus licheni o mis (Wang e al., 2019). Also, he
ansc ip ome o Esche ichia coli agains M. xan hus in liquid media
has been analyzed, and he esul s showed ha he p esence o
he p eda o caused widesp ead induc ion in gene exp ession and
en ichmen o se e al pa hways including ibosome p oduc ion,
lipopolysaccha ide (LPS) biosyn hesis, oxida i e phospho yla ion,
p oduc ion o an ibio ics and seconda y me aboli es, ene gy and
ca bon me abolism, and i amin and amino acid me abolism.
Howe e , only he pa hway in ol ed in glyce ophospholipid
me abolism was down- egula ed (Li ings one e al., 2018).
In he cu en s udy, we ha e analyzed he de ensome o he
p ey S. melilo i agains a ack by M. xan hus.S. melilo i is an
alphap o eobac e ium ha es ablishes ni ogen- ixing symbiosis
wi h legumes, he eby con ibu ing o he e ili y o soils. This
soil bac e ium can also exis as a ee-li ing o ganism in na u al
en i onmen s, whe e i mus adap o di e se nu ien a ailabili y
condi ions, and compe e wi h o he neighbo ing mic obes,
including p eda o s such as M. xan hus. These compe i i e
in e ac ions will a ec no only he s uc u e o he soils, bu also,
ul ima ely, will a ec hei e ili y.
Ou esul s e eal ha he con ac wi h M. xan hus induces
in S. melilo i de ense and/o adap a ion genes ha a ec cen al
pa hways, such as p o ein biosyn hesis and sec e ion. The
econs uc ion o o he up- egula ed pa hways seems o indica e
ha S. melilo i no only p o ec s i sel agains p eda o a ack
in a passi e way by modi ying he cell en elope, bu also eac s
ac i ely by p oducing H2O2o o maldehyde. The induc ion o
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genes ela ed o i on up ake indica e ion compe i ion. This esea ch
comple es he s udy on ansc ip omic changes unde gone in bo h
pa ne s du ing he M. xan hus-S. melilo i in e ac ion and d aws
a pano amic iew o he mechanisms and pa hways in ol ed in
a ack, de ense, and compe i ion.
2. Ma e ials and me hods
2.1. Media, bac e ial s ains and g ow h
condi ions
Sino hizobium melilo i Rm1021 (Meade and Signe , 1977) was
used as p ey, whe eas M. xan hus DK1622 (Kaise , 1979) was used
as p eda o . T yp one yeas (TY) solid and liquid media (Be inge ,
1974) we e used o main enance and g ow h o S. melilo i. CTT
solid and liquid media (Hodgkin and Kaise , 1977) we e used o
g ow M. xan hus, and o he p eda ion expe imen s. Solid media
con ained 1.5% Bac o-Aga (Di co, Le Pon de Claix, F ance), and
liquid cul u es we e incuba ed wi h igo ous shaking a 30◦C.
2.2. P epa a ion o p ey cells and
co-cul u e o p ey and p eda o y cells
Sino hizobium melilo i was g own in TY b o h o an op ical
densi y a 600 nm (OD600) o 1 and hen dilu ed using he same
b o h o a inal OD600 o 0.2. Twen y 10-µl d ops o he dilu ed
cul u e we e deposi ed on he su ace o CTT aga pla es o each
eplica e and incuba ed a 30◦C o 24 h. A e ha ime, S. melilo i
cells om wo eplica es we e ha es ed om pla es o ob ain
= 0 p ey samples (samples Sm_ 0). Then, o ob ain samples o
p eda o y and p ey in e ac ing cells, 10-µl d ops o M. xan hus,
g own in CTT liquid media o an OD600 o 1 and concen a ed
in TM bu e [10 mM T is-HCl (pH 7.6), 1 mM MgSO4] o a
inal OD600 o 15, we e deposi ed on op o each o he hizobial
colonies o a subse o he pla es (samples Mx_Sm). Ano he subse
o samples o S. melilo i was kep g owing alone (samples Sm).
Two eplica es om each o he wo condi ions (p eda o /p ey
co-cul u e and pu e cul u e o S. melilo i) we e ha es ed om
pla es a e 2 and 6 h o incuba ion. Pelle s om each sample we e
esuspended in 0.5 ml o RNA P o ec Bac e ia Reagen (Qiagen,
Hilden, Ge many), incuba ed a oom empe a u e o 5 min, and
cen i uged a 5,000 g o 10 min (4◦C). Nex , pelle s we e s o ed
a −80◦C.
2.3. RNA ex ac ion
To pu i y RNA, cells we e lysed o 10 min a oom
empe a u e wi h lysozyme and p o einase K [250 µl o 3 mg/ml
lysozyme (Roche Diagnos ic, Mannheim, Ge many) and 0.4 mg/ml
p o einase K (Ambion, Ca lsbad, CA, Uni ed S a es)] p epa ed in
TE bu e [10 mM T is-HCl; 1 mM e hylenediamine e aace ic acid
(EDTA), pH 8.0]. RNeasy Mini Ki (Qiagen, Hilden, Ge many) was
used o RNA ex ac ion [ca ying ou on-column DNase diges ion
wi h he RNAse- ee DNase se (Qiagen, Hilden, Ge many)],
elu ing each sample in 50 µl o RNase- ee wa e .
2.4. Lib a y p epa a ion, sequencing, and
global ansc ip omic da a analysis
To al RNA samples we e p ocessed by No ogene [No ogene
Eu ope, Camb idge, Uni ed Kingdom], including RNA deple ion
om o al RNA samples wi h he Illumina Ribo-Ze o Plus RNA
Reduc ion Ki (Illumina, Inc.). Remaining RNA was p ocessed
acco ding o he p ocedu es desc ibed in Pé ez e al. (2022).
On a e age, 18.81 million aw eads and a co e age o 421.75x
we e ob ained. A e emo ing eads con aining adap e s, low
quali y eads and/o eads wi h mo e han 10 pe cen unce ain
nucleo ides, he genome co e age a ied om 355.53x o 494.52x
(median o 415.18x), which p o ide an excellen co e age o
he mRNA ac ion.
To acili a e compa ison analyses wi h o he S. melilo i
ansc ip omes we used he old nomencla u e (SMa_, SMb_
o SMc_), since hese iden i ie s a e commonly used in he
li e a u e. Howe e , he co esponding new localize s SM_RS a e
also indica ed in he ables. To deciphe he de ensome, we ha e
conside ed all he up- and down- egula ed ansc ip s wi h | Log2
Fold Change| >0 and padj <0.05. I mus be aken in o accoun
ha p eda ion is a mul i ace ed p ocess, and any change could be
o in e es as we ha e demons a ed in he case o M. xan hus
p eda osome (Pé ez e al., 2022). The adap a ion and de ense
mechanisms expec ed may no be e y d as ic and, o his eason,
ou esea ch has been ocused on hose ou es in which he e
is gene en ichmen o in hose pa hways in which many o he
genes in ol ed a e up o down egula ed in he p esence o he
p eda o . In all igu es and ables he Log2 Fold Change is speci ied
and hose Log2 Fold Change >1 o Log2 Fold Change <−1
a e highligh ed. Fo esea che s in e es ed in his ansc ip omics,
all da a including eads, agmen s pe kilobase o ansc ip
pe million agmen s mapped (FPKMs) and Log2 Fold Change
a e a ached as Supplemen a y ma e ial. Howe e , o imp o ed
con idence, | Log2 Fold Change| >1 has been used in compa isons
wi h o he ansc ip omes, and his h eshold is also indica ed in all
he igu es and ables.
3. Resul s and discussion
3.1. O e iew o he ansc ip omic
esponse o S. melilo i on p eda o y
co-cul u es
T ansc ip ional changes in S. melilo i in esponse o he
p eda o y a ack by M. xan hus was in es iga ed by using he RNA-
seq echnology. The M. xan hus-S. melilo i co-cul u e condi ions
and he p epa a ion o lib a ies ha e been p e iously desc ibed in
de ail (Pé ez e al., 2022). We ocused ou s udy on wo poin s in
ime a e con ac : a 2 and 6 h, a pe iod o ime du ing which he
p ey has o adap i s me abolism and s uc u es o he bio ic s ess
ha ep esen s he p eda o y a ack. Following he same p ocedu e
used o elucida e he M. xan hus p eda osome (Pé ez e al., 2022),
i e cDNA lib a ies we e cons uc ed o analyze he S. melilo i
de ensome: Sm_ 0: S. melilo i alone a ime 0 h; Sm_ 2: S. melilo i
alone collec ed a e 2 h on solid CTT medium; Sm_ 6: S. melilo i
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alone g own o 6 h on solid CTT medium; Mx_Sm 2: cells
collec ed a e 2 h o in e ac ion be ween M. xan hus and S. melilo i;
and Mx_Sm 6: cells ha es ed a e 6 h o he M. xan hus-S. melilo i
in e ac ion. F om now on, he e ms 2 and 6 will be used o e e
o esul s ob ained a 2 and 6 h o he co-cul u es o M. xan hus
and S. melilo i (Mx_Sm 2 and Mx_Sm 6, espec i ely) compa ed o
hei espec i e con ols (p ey cells in pu e cul u e a 2 o 6 h: Sm_ 2
and Sm_ 6, espec i ely).
The o al numbe o aw eads om each sample, he
clean eads (ob ained a e emo al o aw eads con aining
adap e s and/o o low quali y eads), he e o s Q20 and
Q30, and he GC con en o he clean eads a e compiled in
Supplemen a y Table 1A.
The Pea son co ela ion coe icien s (R2) be ween eplica es
we e sa is ac o y in all samples (≥0.91) (Supplemen a y
Figu e 1A). The p incipal componen analysis (PCA) showed
ha genes ob ained om he same condi ion clus e oge he and
genes om di e en nu i ional s ages and imes clus e sepa a ely
as expec ed (Supplemen a y Figu e 1B). The RNA mean eads
we e no malized o FPKM alues (Supplemen a y Table 1B).
The FPKM densi y dis ibu ions and he iolin diag ams showing
simila gene exp ession le els a e depic ed in Supplemen a y
Figu es 1C, D, espec i ely.
The olcano plo s we e cons uc ed by using he ansc ip s o
co-cul u es a 2 and 6 h e sus hei espec i e con ols il e ed
by hei old changes (| Log2 Fold Change| >0) and padj <0.05
(Figu es 1A, B).
Including no el genes and sRNAs (i.e., non-coding RNAs o
50–500 n leng h), 1,772 and 2,081 ansc ip s we e di e en ially
egula ed in S. melilo i in esponse o p eda o a ack a 2 and 6 h,
espec i ely (Figu es 1A, B and Supplemen a y Tables 1C, D).
Conce ning he di ec ion o ansc ip egula ion, 806 ansc ip s
(45.5%) we e up- egula ed and 966 (54.5%) down- egula ed a
2 h, while 1,108 (53.2%), and 973 (46.8%) ansc ip s we e up-
and down- egula ed, espec i ely, a 6 h. O e all, hese da a
indica e ha 26.5% (2 h) and 31.2% (6 h) o he S. melilo i
ansc ip ome esponded o p eda o a ack. This ep esen s a
change in ansc ip ional ac i i y no ably g ea e han ha obse ed
in he p eda o unde he same expe imen al condi ions (Pé ez
e al., 2022). These esul s a e in ag eemen wi h hose ob ained
du ing p eda ion on E. coli, whe e i was also ound ha p eda ion
caused a much mo e p onounced esponse in he p ey han in he
p eda o (Li ings one e al., 2018), indica ing a s ong adap i e,
compe i i e and/o de ensi e esponse igge ed by he p esence
o he p eda o . Fo u he analyses o he de ensome in his
s udy, no el genes and sRNAs we e no conside ed, lea ing 1,361
and 1,818 ansc ip s as di e en ially egula ed a 2 and 6 h,
espec i ely. These genes ha e been o ganized in 973 up- egula ed
and 1,139 down- egula ed genes iden i ied a 2 h and/o 6 h in
Supplemen a y Tables 2A, B.
We in es iga ed whe he p eda ion al e s gene exp ession
in S. melilo i in a eplicon-biased ashion. The S. melilo i
Rm1021 genome (6.69 Mb in size) is composed o h ee la ge
eplicons: a ch omosome (3.65 Mb) and wo megaplasmids, pSymA
(1.35 Mb), and pSymB (1.68 Mb) ha con ain 54, 21, and 25%
o he o al anno a ed genes (Galibe e al., 2001). We ound
ha genes di e en ially egula ed du ing p eda ion we e no
p opo ionally dis ibu ed among he eplicons. Ins ead, hey we e
biased owa d he ch omosome (70.6%), whe eas only 9.6 and
FIGURE 1
Di e en ial gene exp ession o Sino hizobium melilo i in esponse
o Myxococcus xan hus p eda ion. Volcano plo s o up- egula ed
and down- egula ed genes du ing he p eda o y in e ac ion a (A) 2
and (B) 6 (2 and 6 h o con ac ). The es ima ed old changes
(x-axis) e sus he minus log10 o he adjus ed p- alues (y-axis)
om DESeq analysis a e shown in he olcano plo s. The signi ican
genes wi h absolu e alues o | Log2 Fold Change| >0 and
padj <0.05 a e depic ed in ed (up- egula ed) o in g een
(down- egula ed). Blue do s indica e non- egula ed genes (NO).
G ay e ical do ed lines indica e ze o- old change.
19.8% o he di e en ially exp essed genes we e associa ed o
pSymA and pSymB, espec i ely (Supplemen a y Figu e 2A). This
ch omosomal bias was mainly caused by genes whose exp ession
was inc eased du ing p eda ion since 83.2% we e loca ed on he
ch omosome wi h only 5.6% on pSymA and 11.2% on pSymB.
In con as , down- egula ed genes we e dis ibu ed mo e e enly,
wi h 59.8% on he ch omosome, 13% on pSymA and 27.2% on
pSymB (Supplemen a y Figu e 2B and Supplemen a y Table 3).
These esul s indica e ha in esponse o p eda o a ack, S. melilo i
ac i a es ch omosomal-encoded unc ions, whe eas he symbio ic
plasmids, especially pSymA, ha e a mino con ibu ion.
To iden i y he main biological p ocesses a ec ed in S. melilo i
du ing p eda ion, wo di e en app oaches we e used. In one
o hem, en ichmen analyses we e ca ied ou using he
associa ed pa hways in he KEGG da abase (Kyo o Encyclopedia
o Genes and Genomes; Kanehisa e al., 2021). The up- egula ed
pa hways in ol ed hose ela ed o ibosome p oduc ion, oxida i e
phospho yla ion, and biosyn hesis o amino acids, seconda y
me aboli es, and co ac o s (Supplemen a y Figu e 3A and
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Supplemen a y Tables 1E, F). The main down- egula ed pa hways
du ing p eda ion we e ela ed o aline, leucine, and isoleucine
deg ada ion, mic obial me abolism in di e se en i onmen s,
ca bon me abolism, quo um sensing and ABC anspo e s
(Supplemen a y Figu e 3B and Supplemen a y Tables 1E, F).
As a complemen a y app oach, unc ional ca ego ies o he 973
up- egula ed (Supplemen a y Table 2A) and he 1,139 down-
egula ed (Supplemen a y Table 2B) genes we e de e mined using
clus e s o o hologous g oups (COGs) acco ding o he genome
sequence anno a ions o he S. melilo i Genome P ojec 1(Table 1).
Many di e en ially egula ed genes a e anno a ed as ei he
exhibi ing pa ial o global homology o genes deposi ed in
he da abases (No in COGs) o ha ing unknown unc ions.
Ne e heless, se e al unc ional ca ego ies could be iden i ied
associa ed o up- egula ed genes (highligh ed in ligh g ay in
Table 1), o o down- egula ed genes (highligh ed in da k g ay
in Table 1). These analyses sugges ha he S. melilo i esponse
o he p eda o y a ack by M. xan hus in ol es he ac i a ion
o p o ein syn hesis and sec e ion, inc eased ene gy gene a ion,
changes in he cell en elope and memb anes, and he s imula ion o
he anspo and me abolism o ino ganic ions and nucleo ides. A
he same ime, ca bohyd a e and lipid anspo and me abolism,
as well as signal ansduc ion mechanisms and cell di ision- ela ed
unc ions a e ep essed du ing p eda ion. Compa ison o he
S. melilo i de ensome ob ained in he p esen s udy wi h ha o
E. coli (Li ings one e al., 2018) e eals some simila i ies bu also
some di e ences. Thus, inc eased p o ein p oduc ion and ene gy
gene a ion, and he biosyn hesis o seconda y me aboli es we e
de ec ed du ing p eda ion on he wo p eys. Howe e , whe eas
ca bon me abolism and glyce ophospholipid me abolism we e up-
and down- egula ed, espec i ely, in E. coli du ing p eda ion, he
opposi e egula ion was ound o he same unc ional ca ego ies
in S. melilo i. I hese di e ences e lec di e en e asion/de ense
s a egies o he p ey o i hey a e he esul o di e en
expe imen al app oaches emains unknown.
3.2. P eda ion on S. melilo i ac i a es
p o ein p oduc ion and sec e ion, a y
acid syn hesis, and ene gy gene a ion
while ep essing a y acid deg ada ion
and ca bohyd a e anspo and
me abolism
A la ge ac ion (88%) o he genes in ol ed in ansla ion
which we e iden i ied as di e en ially egula ed in he
ansc ip ome p o ile exhibi ed inc eased exp ession di e en ially
exp essed genes in ol ed in ansla ion iden i ied in ou
ansc ip ome exhibi ed inc eased exp ession du ing p eda ion
compa ed wi h con ol condi ions (Table 1). O he 95 up- egula ed
genes ela ed o ansla ion, 28 code o ibosomal p o eins and
p o eins in ol ed in ibosome ma u a ion and modi ica ion, and
33 o aminoacyl- RNA syn he ases and p o eins ela ed wi h RNA
modi ica ion. In addi ion, genes pu a i ely coding o ansla ion
ini ia ion (in A,in B,in C) and elonga ion ( s ,e p) ac o s, as well
1h ps://ian . oulouse.in a. /bac e ia/anno a ion/cgi/ hime.cgi
as p obable pep ide chain elease ac o s (p A,p B,p C) we e
also up- egula ed, s ongly sugges ing he ac i a ion o p o ein
syn hesis du ing p eda ion. Hence, ansla ion is a biological
p ocess ac i a ed du ing p eda ion in di e en p ey (Li ings one
e al., 2018), bu also in he p eda o (Pé ez e al., 2022).
In ag eemen wi h inc eased p o ein syn hesis and he
consequen g ea e demand o amino acids, 111 genes in ol ed
in amino acid anspo and me abolism we e also up- egula ed.
Mo eo e , genes coding o he ATP-dependen chape one olding
sys em DnaK/DnaJ/G pE exhibi ed inc eased exp ession. This
sys em plays a c ucial ole in mic obial p o eos asis unde bo h
no mal and s ess condi ions by assis ing he olding o newly
syn hesized polypep ides, and o mis olded o agg ega ed p o eins
(Win e and Jakob, 2004;Ba io e al., 2020). Recen ly, S. melilo i
DnaJ has been shown o pa icipa e in ole ance o di e en s esses
(B i o-San ana e al., 2023). Besides assis ing p o ein olding, he
DnaK chape one also acili a es p o ein a ge ing o memb anes
and p o ein ansloca ion (Ba io e al., 2020). In e es ingly, we
ound ha he majo i y o genes in ol ed in in acellula a icking
and sec e ion iden i ied as di e en ially exp essed du ing p eda ion
we e also up- egula ed (Table 1), sugges ing inc eased p o ein
sec e ion. This was he case o se e al genes ela ed o he Sec
sys em, which is esponsible o he inse ion, ansloca ion, and
sec e ion ac oss he memb ane o un olded polypep ides, which
ca y a emo able N- e minal signal sequence. Thus, genes coding
o he memb ane-embedded SecYEG anslocon, he SecA ATPase
and pilo ing ac o s such as he signal ecogni ion pa icle (SRP) F h
o he SecB chape one, which main ain newly syn hesized p o eins
in an un olded con o ma ion and d i e hem o he memb ane
(Papanikou e al., 2007), exhibi ed inc eased exp ession du ing
p eda ion. In con as o he Sec anslocon, he Ta sys em is
esponsible o expo ing p e iously olded p o eins which ha e a
pa icula signal pep ide con aining a ecognizable win-a ginine
mo i (Picke ing and O esnik, 2010;Palme and Be ks, 2012). These
p o eins seem also o be ac i ely expo ed in S. melilo i du ing
p eda ion, as sugges ed by he up- egula ion o he a A gene
coding o a Ta A/E anslocase homolog. The up- egula ion o
p o ein sec e ion sys ems has also been epo ed in myxobac e ia,
whe e i has been associa ed o he sec e ion o ac o s equi ed o
p eda ion o bac e ia and ungi (Li e al., 2019;Pé ez e al., 2022). In
he case o he p ey, inc eased p o ein sec e ion could be equi ed o
main ain bac e ial cell en elope p o ein complexes, whose in eg i y
migh be damaged du ing p eda o y a ack.
Many genes coding o he ype II a y acid syn hase (FAS
II) sys em, which is esponsible o FA syn hesis, we e ound o
be up- egula ed in S. melilo i du ing p eda ion by M. xan hus
(Figu es 2A,2C). These include genes coding o mos o he
enzymes in ol ed in he ini ia ion phase and elonga ion cycles
o FA chains (López-La a and So o, 2018), as well as some
pa alogous genes. Ac i a ion o FA syn hesis is also sugges ed by
he inc eased exp ession o acpS (smc02654), he holo-acyl-ca ie
p o ein (ACP) syn hase, which ans e s he 4’-phosphopan e heine
om coenzyme A (CoA) o apo-ACP, he eby con e ing i o he
unc ional holo-ACP o which acyl in e media es can be bound.
The up- egula ion o abA and abB equi ed o unsa u a ed
FA syn hesis, oge he wi h ha o abI and abF, sugges s ha
he syn hesis o bo h sa u a ed and unsa u a ed FAs is inc eased
du ing p eda ion (López-La a and So o, 2018). In con as , FA
deg ada ion dec eased du ing p eda o y a ack, as indica ed by he
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down- egula ion o se e al genes equi ed o his p ocess. Among
hem, genes coding o p o eins in ol ed in he up ake o long-
chain FAs ( adL), genes in ol ed in he ac i a ion o di e en FAs
wi h CoA ( adD,ma B,smc00261,smb20650), as well as genes o
he smc02229- adA- adB ope on, which mos likely code o he
enzymes equi ed o he di e en s eps in he β-oxida ion cycle,
a e ound (Figu es 2B,2D). Dec eased FA deg ada ion, oge he
wi h inc eased FA biosyn hesis, could indica e ha S. melilo i
equi es FAs o a unc ion o he han ene gy p oduc ion, pe haps
o building and emodeling he cell memb ane in esponse o
p eda o y a ack (see sec ion “3.4. D as ic changes in he S. melilo i
cell en elope du ing p eda ion”). Lipid biosyn hesis is also ac i a ed
TABLE 1 Func ional ca ego ies o genes di e en ially exp essed in
Sino hizobium melilo i in esponse o p eda ion by Myxococcus xan hus.
Gene ca ego y Numbe o
up- egula ed
genes(1)
Numbe o
down- egula ed
genes
No in COGs(2)123 (43.5%) 160 (56.5%)
Func ion unknown 32 (28.3%) 81 (71.7%)
T ansla ion 95 (88%) 13 (12%)
In acellula a icking and
sec e ion
14 (87.5%) 2 (12.5%)
Cell wall/memb ane biogenesis 52 (62.7%) 31 (37.3%)
Ino ganic ion anspo and
me abolism
55 (61%) 35 (39%)
Nucleo ide anspo and
me abolism
36 (61%) 23 (39%)
Seconda y me aboli es
biosyn hesis, anspo , and
ca abolism
17 (50%) 17 (50%)
T ansc ip ion 49 (46.7%) 56 (53.3%)
Pos ansla ional modi ica ion,
p o ein u no e , chape ones
37 (46.2%) 43 (53.8%)
De ense mechanisms 10 (43.5%) 13 (56.5%)
Gene al unc ion p edic ion only 76 (43.2%) 100 (56.8%)
Amino acid anspo and
me abolism
111 (42.7%) 149 (57.3%)
Cell mo ili y 11 (42.3%) 15 (57.7%)
Replica ion, ecombina ion, and
epai
19 (42.2%) 26 (57.8%)
Ene gy p oduc ion and con e sion 63 (40.9%) 91 (59.1%)
Coenzyme anspo and
me abolism
26 (40%) 39 (60%)
Lipid anspo and me abolism 33 (37.1%) 56 (62.9%)
Signal ansduc ion mechanisms 16 (35.6%) 29 (64.4%)
Cell cycle con ol, mi osis and
meiosis
7 (35%) 13 (65%)
Ca bohyd a e anspo and
me abolism
44 (23.2%) 146 (76.8%)
(1)Pe cen o up- egula ed genes wi hin each gene ca ego y is shown in pa en hesis.
Func ional ca ego ies in which mo e han 60% o he genes di e en ially egula ed show
inc eased o educed exp ession du ing p eda o a ack a e shown in ligh o da k
g ay, espec i ely.
(2)COG, clus e o o hologous g oups.
in M. xan hus du ing p eda ion, mos likely o change he lipid
composi ion o he cell en elope and o syn hesize new seconda y
me aboli es ha will con ibu e o kill p ey. Howe e , and in
con as o i s p ey, genes coding o enzymes in ol ed in he β-
oxida ion cycle a e up- egula ed indica ing an inc eased ene gy
demand du ing p eda o y a ack (Pé ez e al., 2022).
Consis en wi h inc eased syn hesis o p o eins and FA, which
a e me abolically demanding p ocesses, he up- egula ion o up
o 63 genes in ol ed in ene gy p oduc ion and con e sion was
de ec ed. These include genes coding o di e en complexes
o he espi a o y chain and associa ed unc ions, such as he
ch omosomal nuo genes ha code o he p o on-pumping NADH:
ubiquinone oxido educ ase (nuoA1B1C1D1E1F1HIJK1LMN),
genes o cy och ome o ubiquinol oxidase (cyoABC), cy och ome c
oxidase (c aCD), cy och ome b ( bcB), and a pu a i e ATP syn hase
(a pBCF2). Inc eased exp ession o genes ha code o di e en
enzymes o he ica boxylic acid cycle was also de ec ed (pdhABC,
lpdA1,sdhABCD, umC,sucA,icd,acnA,mdh).
Simila o genes in ol ed in FA deg ada ion, he majo i y o
he genes belonging o he ca ego y o ca bohyd a e anspo and
me abolism (76.8%) we e down- egula ed in he ansc ip ome.
Genes coding o di e en suga ABC anspo e s (ugpABC,
cAK, haST), glucoly ic enzymes (pgm,pgi,pgiA1,cbbA,cbbA2,
piA1,gap,pgk,pykA), enzymes o he pen ose phospha e
pa hway (cbbT,gnd,eda2, bsK, uD1), and enzymes in ol ed
in glycogen (glgC,glgA1) and poly-3-hyd oxybu y a e (phbABC)
syn hesis, showed educed exp ession compa ed wi h con ol
condi ions. Dis up ion o he glycogen syn hase gene glgA1
esul ed in dec eased polyhyd oxybu y a e (PHB) le els and
inc eased EPS le els compa ed o he wild ype (D’Alessio e al.,
2017). In s a ing condi ions, he use o glycogen could be
an al e na i e ene gy sou ce. In a o o his hypo hesis is he
ac ha gene glgX1, in ol ed in glycogen-deb anching, is up-
egula ed.
3.3. Inc eased exp ession o genes
ela ed o i on and phospho us
s a a ion esponses
Ino ganic anspo and me abolism was a unc ional ca ego y
ha showed en ichmen in up- egula ed genes iden i ied in he
esponse o p eda o y a ack (Table 1). Wi hin his ca ego y,
i was ema kable he up- egula ion o many genes in ol ed
in i on up ake and me abolism, including genes in ol ed in
he syn hesis ( hbABDEF and sma2339) and up ake ( h A,
h X) o he side opho e hizobac in 1021 (Lynch e al., 2001).
Addi ional genes ela ed wi h i on and wi h inc eased exp ession
a e hose in ol ed in he up ake o heme and hyd oxama e
side opho es [shmR,hmuSTV, huA2/ oxA, huA3, huP,sma1742
epG (sma1742), epB (sma1746)], he exbD gene encoding one
o he componen s o he TonB-ExbB-ExbD complex, which
p o ides he ene gy o he anspo o heme and side opho e-
media ed i on anspo ac oss he ou e memb ane, he huF
gene coding o e ioxamine B educ ase, o genes o pu a i e
Fe3+ABC anspo e s [i p6C (smb21429) and i p6B (smb21430)]
(Fabiano and O’B ian, 2012;O’B ian, 2015) (Figu es 3A–C).
Genes coding o egula o y p o eins in ol ed in i on homeos asis
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FIGURE 2
Changes in a y acids (FA) me abolism in S. melilo i in co-cul u es wi h Myxococcus xan hus a e 2 and 6 h o con ac ( 2 and 6). (A) Up- egula ion
o genes in ol ed in FA biosyn hesis. ML, memb ane lipids (see Figu e 4 o mo e in o ma ion). (B) Down- egula ion o he genes esponsible o he
β-oxida ion deg ada i e pa hway. Those genes (up o down- egula ed) wi h demons a ed ac i i y in he li e a u e a e indica ed by hei names (see
ex o de ails), while pa alogous genes ound in he KEGG da abase and ha a e also di e en ially exp essed a e ep esen ed by hei gene
iden i ie s. (C,D) Hea maps o he genes in ol ed in FA biosyn hesis and FA β-oxida ion, espec i ely. Red o g een edges indica e genes wi h | Log2
Fold Change| >1, and do ed edges indica e no di e en ially exp essed genes a he indica ed ime.
(Supplemen a y Figu es 4A, B and Supplemen a y Tables 4A, B)
we e also di e en ially exp essed du ing p eda o y a ack. Genes
coding o HmuP, which con ols hemin acquisi ion, Rh A, which
con ols bo h he syn hesis and he up ake o hizobac in 1021,
and he i on egula o I we e up- egula ed, whe eas he gene
coding o he hizobial i on egula o A (Ri A) was down-
egula ed (Ama elle e al., 2010;O’B ian, 2015). I senses i on
indi ec ly h ough he s a us o heme biosyn hesis (Figu e 3D).
Unde i on-limi ing condi ions, I ep esses genes encoding
p o eins ha unc ion unde i on-su icien condi ions, including
i A (O’B ian, 2015). Ri A is an i on-sul u p o ein ha ac s as a
ep esso o i on-up ake unde i on- eple e condi ions (Chao e al.,
2005;Pellice Ma inez e al., 2017;Figu e 3B). The e o e, da a
ob ained he e indica e ha S. melilo i is expe iencing i on-limi ing
condi ions du ing co-cul u e wi h he myxobac e ial p eda o ,
simila o he si ua ion p e iously epo ed o S. coelicolo (Lee
e al., 2020) and Pseudomonas pu ida (Akba and S e ens, 2021).
Up- egula ion o he i on-up ake machine y was also obse ed
in M. xan hus du ing co-cul u e wi h S. melilo i (Pé ez e al.,
2022), sugges ing ha bo h p eda o and p ey a e compe ing
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o i on. I would be in e es ing o es whe he inc eased
hizobac in 1021 p oduc ion in S. melilo i Rm1021 enhances
hizobial esis ance o myxobac e ial p eda ion as shown o
P. pu ida su i o s ha o e p oduce he side opho e pyo e dine
(Akba and S e ens, 2021).
I is known ha he me abolism o i on and manganese
a e in e ela ed (O’B ian, 2015). Manganese can eplace i on
in many enzymes. Howe e , and despi e expe iencing low
i on concen a ions, he exp ession o he mn ABCD ( o me ly
si ABCD) genes coding o a manganese ABC anspo e (Pla e o
e al., 2003) and ha o i s egula o Mu we e down- egula ed
du ing p eda ion (Figu e 3E).
Se e al genes ela ed wi h phospho ous up ake and me abolism
we e up- egula ed in he de ensome o S. melilo i, such as hose
coding o phospha e ABC- ype anspo e s (phoCDE,ps A),
phosphona e me abolism (phnGHIJ), alkaline phospha ase (phoX),
polyphospha e kinase (ppk), as well as genes coding o egula o y
p o eins ha a e c ucial o he main enance o cellula phospha e
homeos asis (phoB and phoU). In iguingly, up o 29 o he up-
egula ed genes in he de ensome we e also ound o be up-
egula ed in he phospha e s a a ion esponse o S. melilo i, wi h
he majo i y o hem (25) belonging o he clus e I o PhoB-
dependen genes induced by phospha e s ess (Supplemen a y
Figu e 4B and Supplemen a y Tables 4C, D;K ol and Becke ,
2004). PhoB is he esponse egula o o he PhoR-PhoB wo-
componen sys em ha con ols a se o genes known as he
Pho egulon, which is in ol ed in cell adap a ion o phospha e
s a a ion. In addi ion o genes ela ed o phospho us up ake and
me abolism, he Pho egulon includes o he genes such as hose
ela ed o phospho us- ee memb ane lipid biosyn hesis (sqdB,
b aAB,olsA,olsB), which we e also up- egula ed du ing p eda o y
a ack. Mo eo e , 29 genes up- egula ed in he de ensome inc eased
hei exp ession le els unde phospha e s a a ion in a PhoB-
independen manne . This g oup includes se e al genes in ol ed
in he syn hesis o he exopolysaccha ide I (EPS I) o succinoglycan
(exoA,exoW,exoV,exoK,exoYF1,exoX) (see also Sec ion “3.4.
D as ic changes in he S. melilo i cell en elope du ing p eda ion”).
The ansc ip ional ac i a ion o low phospha e- esponsi e genes,
including membe s o he Pho egulon, could be in e p e ed as
S. melilo i cells acing phospha e-limi ing condi ions. Howe e , his
is di icul o belie e conside ing ha he medium used in he
expe imen al se up is a phospha e- ich medium and ha phoU
is ansc ip ionally up- egula ed. I has been sugges ed ha he
egula o y p o ein PhoU esponds o ele a ed phospha e le els by
signi ican ly dec easing he phospha e anspo o Ps SCAB o
p e en phospha e oxici y (diCenzo e al., 2017). Mo eo e , he
ac i a ion o EPS I p oduc ion is known o occu unde high-
phospha e condi ions (Mend ygal and González, 2000;Acos a-
Ju ado e al., 2021). The e o e, and mos likely, he up- egula ion
o low phospha e- esponsi e genes in he de ensome canno be
he di ec esul o a phospho ous de iciency. In E. coli, PhoB is
no only ac i a ed by low phospha e, bu also by cell en elope
s ess (Choudha y e al., 2020). I is no un easonable o hink ha
compounds and hyd oly ic enzymes sec e ed by M. xan hus du ing
p eda o y a ack dis up di e en componen s o he S. melilo i
cell en elope, causing cell en elope s ess. Ac i a ion o he PhoB
egula o by he cell en elope s ess caused du ing p eda o y a ack
and/o any o he mechanism, oge he wi h he known o e lap and
in e ac ion o he Pho egulon wi h se e al o he con ol ci cui s
(Yuan e al., 2006), could explain he di e en ial exp ession o low
phospha e-induced genes du ing myxobac e ial p eda ion.
3.4. D as ic changes in he S. melilo i cell
en elope du ing p eda ion
The bac e ial cell en elope is a complex s uc u e ha p o ides
s uc u al in eg i y and p o ec s he cy oplasm om changes in he
su ounding en i onmen . As a dide m bac e ium, he hizobial
cell en elope consis s o h ee laye s: he cy oplasmic o inne
memb ane (IM), a hin pep idoglycan cell wall, and he ou e
memb ane (OM) con aining LPS in he ou e lea le . In addi ion,
hizobia p oduce di e en su ace polysaccha ides (SPSs) ha
se e di e en unc ions du ing he ee-li ing and symbio ic
li es yles o hese bac e ia (Acos a-Ju ado e al., 2021). Main enance
o cell en elope in eg i y is essen ial o iabili y, and bac e ia
ha e de eloped egula o y mechanisms o de end om en elope
dis u bance. Conside ing he epibio ic p eda o y s a egy employed
by M. xan hus, i is no su p ising ha many S. melilo i genes ela ed
wi h he di e en cell en elope s uc u es a e ansc ip ionally
modula ed in esponse o p eda o a ack. Below, we p o ide
in e p e a ion o he ansc ip ional changes de ec ed in genes
associa ed o di e en cell en elope s uc u es.
In S. melilo i, he exo/exs and wgx ( o me ly exp) genes a e
esponsible o he p oduc ion o wo di e en kinds o acidic
exopolysaccha ides (EPSs): EPS I o succinoglycan, and EPS II o
galac oglucan (Becke e al., 2002). The egula ion o hese EPSs is
e y complex, and se e al en i onmen al condi ions and egula o y
p o eins ha e been in ol ed in hei con ol (Ba ne and Long,
2018). As p e iously men ioned, se e al exo/exs genes in ol ed in
he syn hesis o EPS I (exoA,exoB,exoQ,exoF1,exoH,exoK,exoX,
exoV,exoY,exoN,exoP,exoW,exoK,exsA,exsI) (Glucksmann e al.,
1993) we e ound o be up- egula ed du ing p eda ion. Conce ning
EPS II, he S. melilo i s ain Rm1021 used in his s udy lacks a
unc ional ExpR, a LuxR- ype egula o ha is equi ed o he
quo um sensing-dependen p oduc ion o high amoun s o EPS
II (Pellock e al., 2002). Ne e heless, he inc eased exp ession o
wgcA (expC), wgdA (expE1) and ha o he wggR (expG) gene
ha codes o he ansc ip ional ac i a o o EPS II- ela ed genes,
sugges s imula ion o EPS II syn hesis. T ansc ip ional ac i a ion
o EPS II- ela ed genes could be media ed by PhoB, which ac i a es
exp ession o wggR (Bahlawane e al., 2008). As discussed in sec ion
“3.3. Inc eased exp ession o genes ela ed o i on and phospho us
s a a ion esponses,” he up- egula ion o EPS I- ela ed genes is
independen o PhoB. In his case, he ansc ip ional ac i a ion
o he exo/exs genes could be media ed by egula o y p o eins and
ci cui s known o in luence EPS I p oduc ion and whose exp ession
was also induced du ing p eda ion. This is he case o mucR
(Bahlawane e al., 2008), he exoS/ch G gene o he ExoR-ExoS-
Ch I sys em (Yao e al., 2004;Wells e al., 2007). and he n Y
and n X genes o he N Y-N X egula o y sys em (Cala a a-
Mo ales e al., 2017). Recen ly, he ac i a ion o he Ch G/ExoS-
Ch I egulon in esponse o cell wall s ess has been sugges ed
o Alphap o eobac e ia (Williams e al., 2022), as well as he co-
o dina ed wo k o he ExoR-ExoS-Ch I and N Y-N X sys ems o
con ol di e en unc ions, including egula ion o he bac e ial cell
en elope (S ein e al., 2021).
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FIGURE 3
I on up ake and hizobac in 1021 biosyn hesis a e induced du ing compe i ion. (A) Sino hizobium melilo i genes in ol ed in side opho e syn hesis
and i on-up ake egula ion ha a e induced a 2 o /and 6. Red and blue ci cles ep esen Fe3+and Fe2+, espec i ely. (B,C) Hea maps o he Ri A
and I dependen genes which a e depic ed in panel (A).(D) Con ol o i on homeos asis by he egula o s: Ri A, Rh A, HmuP and I . Ri A is a
(4Fe–4S) clus e con aining p o ein which ep esses many genes in ol ed in i on up ake unde i on- eple e condi ions. The manganese esponsi e
Fu -like ep esso , Mu , con ols manganese up ake. Bo h global egula o y p o eins a e down- egula ed du ing p eda ion, indica ing a mechanism
o he con ol o i on homeos asis by manganese as has been sugges ed o o he bac e ia (see ex o de ails). (Fe–S) clus e s a e depic ed as blue
and yellow ci cles. B own ci cles ep esen Mn2+. A ows and unca ed lines indica e posi i e and nega i e egula ion, espec i ely. OM, ou e
memb ane; IM, inne memb ane. (E) Down- egula ion o i A,mu and Mu -dependen genes (see ex , and Supplemen a y Tables 4A, B o de ails).
In addi ion o EPS I and EPS II, he p oduc ion o o he SPSs
seems o be ac i a ed in S. melilo i du ing p eda ion. This is he case
o he low-molecula mass K polysaccha ide (KPS) (F aysse e al.,
2005), and an as-ye -uncha ac e ized SPS ha con e s esis ance
o he an ibio ic phazolicin (PHZ) and p o isionally named PPP
(PHZ-p o ec ing polysaccha ide) (T a in e al., 2023). Se e al
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