Development versus predation: Transcriptomic changes during the lifecycle of Myxococcus xanthus

F on ie s in Mic obiology 01 on ie sin.o g
De elopmen e sus p eda ion:
T ansc ip omic changes du ing
he li ecycle o Myxococcus
xan hus
JuanaPé ez
†, F anciscoJa ie Con e as-Mo eno
†,
JoséMuñoz-Do ado and Au elioMo aleda-Muñoz *
Depa amen o de Mic obiología, Facul ad de Ciencias, Uni e sidad de G anada, G anada, Spain
Myxococcus xan hus is a mul icellula bac e ium wi h a complex li ecycle.
I is a soil-dwelling p eda o ha p eys on a wide a ie y o mic oo ganisms
by using a g oup and collabo a i e epibio ic s a egy. In he absence o
nu ien s his myxobac e ium en e s in a unique de elopmen al p og am by
using sophis ica ed and complex egula o y sys ems whe e mo e han 1,400
genes a e ansc ip ional egula ed o guide he communi y o agg ega e in o
mac oscopic ui ing bodies illed o en i onmen ally esis an myxospo es.
He ein, weanalyze he p eda osome o M. xan hus, ha is, he ansc ip omic
changes ha he p eda o unde goes when encoun e s a p ey. This s udy has
been ca ied ou using as a p ey Sino hizobium melilo i, a ni ogen ixing bac e ia
e y impo an o he e ili y o soils. The ansc ip ional changes include
up egula ion o genes ha help he cells o de ec , kill, lyse, and consume
he p ey, bu also down egula ion o genes no equi ed o he p eda o y
p ocess. Ou esul s ha e shown ha , as expec ed, many genes encoding
hyd oly ic enzymes and enzymes in ol ed in biosyn hesis o seconda y
me aboli es inc ease hei exp ession le els. Mo eo e , i has been ound ha
he p eda o modi ies i s lipid composi ion and o e p oduces side opho es
o ake up i on. Compa ison wi h de elopmen al ansc ip ome e eals ha
M. xan hus down egula es he exp ession o a signi ican numbe o genes
coding o egula o y elemen s, many o which ha e been demons a ed o
bekey elemen s du ing de elopmen . This s udy shows o he i s ime a
global iew o he M. xan hus li ecycle om a ansc ip ome pe spec i e.
KEYWORDS
Myxococcus xan hus, Sino hizobac e ium melilo i, bac e ial p eda ion,
de elopmen , ansc ip ome, p eda osome
In oduc ion
P eda o y bac e ia use o he li ing bac e ia o ungi as ood sou ces, o which hey
ac i ely hun o kill hem and consume hei mac omolecules as nu ien s. These small
p eda o s a e widely dis ibu ed in many na u al and a i icial en i onmen s whe e
hey play impo an oles in main aining mic obial di e si y and shaping ecosys ems
TYPE O iginal Resea ch
PUBLISHED 26 Sep embe 2022
DOI 10.3389/ micb.2022.1004476
OPEN ACCESS
EDITED BY
Zhaomin Yang,
Vi ginia Tech,
Uni edS a es
REVIEWED BY
Beiyan Nan,
Texas A&M Uni e si y, Uni edS a es
Dan Wall,
Uni e si y o Wyoming,
Uni edS a es
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
SPECIALTY SECTION
This a icle was submi ed o
Mic obial Physiology and Me abolism,
a sec ion o he jou nal
F on ie s in Mic obiology
RECEIVED 27 July 2022
ACCEPTED 08 Sep embe 2022
PUBLISHED 26 Sep embe 2022
CITATION
Pé ez J, Con e as-Mo eno FJ,
Muñoz-Do ado J and
Mo aleda-Muñoz A (2022) De elopmen
e sus p eda ion: T ansc ip omic changes
du ing he li ecycle o Myxococcus
xan hus.
F on . Mic obiol. 13:1004476.
doi: 10.3389/ micb.2022.1004476
COPYRIGHT
© 2022 Pé ez, Con e as-Mo eno, Muñoz-
Do ado and Mo aleda-Muñoz. This is an
open-access a icle dis ibu ed unde he
e ms o he C ea i e Commons A ibu ion
License (CC BY). The use, dis ibu ion o
ep oduc ion in o he o ums is pe mi ed,
p o ided he o iginal au ho (s) and he
copy igh owne (s) a e c edi ed and ha
he o iginal publica ion in his jou nal is
ci ed, in 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.
Pé ez e al. 10.3389/ micb.2022.1004476
F on ie s in Mic obiology 02 on ie sin.o g
(Chauhan e al., 2009; Chen e al., 2011; G i in e al., 2013;
Kandel e al., 2014; Johnke e al., 2017). In he las yea s, bac e ial
p eda o s ha e been ecei ing subs an ial ecogni ion and a e
a ac ing he a en ion o many esea ch g oups because o hei
po en ial applica ions as al e na i e he apies, in which whole
cells o p eda o s can beused as weapons o kill o con ol he
g ow h o o he esis an bac e ia; ha is, hei use in bac e ial
he apy as “li ing an ibio ics” o as biological esou ces p oducing
inno a i e an imic obials and o he bioac i e p oduc s (Pé ez
e al., 2016, 2020).
Bac e ial p eda o s can beg ouped in o wo main hun ing
ca ego ies: endobio ic and epibio ic (Pé ez e al., 2016). Howe e ,
he e a e some bac e ial p eda o s ha , al hough can ac as soli a y
epibio ic hun e s, in na u e p e e ably a ack o ming mul icellula
g oups whe e indi idual cells coope a e wi hin he communi y by
sha ing mix u es o di usible hyd oly ic enzymes and seconda y
me aboli es ha kill and decompose he p ey be o e consuming
he eleased nu ien s (Pé ez e al., 2020; Thie y and Kaime ,
2020). This la e s a egy is used by myxobac e ia, which a e
abundan in soils and ha e been ecen ly desc ibed as a key axon
in he soil ood webs (Pe e s e al., 2021). Myxococcus xan hus is
he mos s udied myxobac e ia, and besides being a p eda o , i is
also a model o ganism o s udying p oka yo ic de elopmen and
bac e ial mul icellula i y (Muñoz-Do ado e  al., 2016). This
bac e ium mo es on solid su aces in a coo dina ed manne , and
o ms dynamic, mul icellula g oups called swa ms, wi hin which
cells in e ac wi h each o he by complex in e -and ex acellula
signaling sys ems. I mo es by using wo di e en locomo ion
sys ems, social mo ili y (S-mo ili y) and ad en u ous mo ili y
(A-mo ili y). In na u al en i onmen s, whe e hey sha e niche
wi h o he mic oo ganisms, M. xan hus swa ms mo e in a
coo dina e way, and when hey ind p ey, he popula ion lyses
hem and abso bs hei nu ien s (Muñoz-Do ado e al., 2016).
Unde nu ien -de icien condi ions, M. xan hus accomplishes he
de elopmen al cycle, whe e housands o o ganized bac e ia
agg ega e o ming mac oscopic s uc u es e med ui ing bodies.
Du ing de elopmen , h ee subpopula ions o cells show di ision
o labo : a small ac ion o cells becomes ound esis an
myxospo es, ano he pa emains as pe iphe al ods, whe eas
mos o he cells die, p obably o p o ide nu ien s ha allow cells
o agg ega e and di e en ia e. When nu i ional condi ions a e
a o able again, he myxospo es in a ui ing body ge mina e and
o igina e a p eda o y swa m (Muñoz-Do ado e al., 2016).
Fo many yea s, myxobac e iologis s ha e concen a ed hei
e o s on he de elopmen al cycle, and a p o ound knowledge has
been accumula ed on he complex egula ion, he in a-and
ex acellula signaling, and he mo ili y mechanisms ha equi es
his mul icellula beha io (Claessen e al., 2014; Cao e al., 2015;
Pé ez e al., 2016). The e a e also many s udies on he use o
myxoba e ia as mic o- ac o ies o new p oduc s (Bade e al.,
2020), and o he esea ch g oups a e s udying complex adap ions
o changing en i onmen s (Muñoz-Do ado e al., 2016; Pé ez
e al., 2018; Padmanabhan e al., 2021). Al hough he i s s udies
on bac e ial p eda o s, mo e han 80 yea s ago, we e ca ied ou
wi h myxobac e ia, only in he las decades his ai has a ac ed
he a en ion o esea che s and some aluable in o ma ion has
been published o elucida e he sys ems and enzymes used by
M. xan hus o con ac and p ey on a b oad a ie y o
mic oo ganisms ( o e iews see Keane and Be leman, 2016; Pé ez
e al., 2016; Thie y and Kaime , 2020).
M. xan hus holds one o he la ges genomes among
p oka yo es, which encodes an uncommon high numbe o
egula o y mechanisms and exhibi s a huge biosyn he ic capaci y
o deg ada i e enzymes and seconda y me aboli es p oduc ion
(Goldman e al., 2006; Pé ez e al., 2008; Muñoz-Do ado e al.,
2019). Al hough he e a e some excellen g oups s udying he
p oduc ion o seconda y me aboli es and new an imic obial
agen s, and se e al bioac i e new p oduc s ha e been isola ed and
cha ac e ized (He mann e al., 2017; Ho mann e  al., 2018;
Bade e  al., 2020), many o he p edic ed biosyn he ic gene
clus e s (BGCs) emain silenced o a e exp essed a low le el
unde he labo a o y condi ions assayed. Consequen ly, mo e
esea ch is needed o exploi mo e e icien ly he eno mous
po en ial o his p eda o (Pé ez e  al., 2020). In his sense,
ansc ip omic s udies by using massi e sequencing echniques
will help o es di e en condi ions ha esemble na u al
en i onmen s o unlock he exp ession o genes coding o
enzymes in ol ed in seconda y me aboli es biosyn hesis ha
could beo pha macological o indus ial in e es . In ac , he
exp ession p o iles o he dynamic ansc ip ome in de elopmen al
condi ions showed ha many o hese BGCs inc ease du ing
de elopmen , p obably o de end spo es inside he ui ing bodies
o o elease nu ien s om p eys o p omo e ge mina ion
(Muñoz-Do ado e al., 2019). Simila ly, elucida ion o M. xan hus
ansc ip omic changes in di e se p eda o y condi ions and wi h
di e en p ey will allow no only o inc ease he exp ession o
c yp ic genes, bu also o assign unc ions o hypo he ical p o eins.
In ou g oup wea e s udying p eda ion o M. xan hus on
o he soil bac e ia ha a e impo an om a bio echnological and/
o ag icul u al poin o iew, such as he an ibio ic p oduce
S ep omyces coelicolo and he legume symbion , ni ogen ixing
Sino hizobium melilo i. Weha e demons a ed ha he p esence
o M. xan hus induces mul icellula i y and o e -p oduc ion o he
blue-colo an ibio ic ac ino hodin on S. coelicolo (Pé ez e al.,
2011). I has also been demons a ed ha du ing co-cul u e o
M. xan hus wi h e e ence labo a o y s ains and ield isola es o
S. melilo i he p eda o exhibi s wo di e en p eda o y s a egies
and ha he exopolysaccha ide galac oglucan (EPS II) is he majo
de e minan o hese pa e ns. This s udy also showed ha A- and
S-mo ili y a e equi ed o e icien p eda ion in he condi ions
assayed (Pé ez e al., 2014). Mo eo e , i has been e ealed ha
coppe plays an impo an ole in M. xan hus-S. melilo i
in e ac ion, inducing in he p ey he biosyn hesis o melanin o
p o ec i sel agains p eda ion (Con e as-Mo eno e al., 2020).
Now, we a e ocused on he elucida ion o he M. xan hus
p eda osome. The p eda osome can be de ined as he
ansc ip omic changes in he p eda o in esponse o he p esence
o he p ey. Wewill use his e m o e e o he up egula ed genes
Pé ez e al. 10.3389/ micb.2022.1004476
F on ie s in Mic obiology 03 on ie sin.o g
ha will help o he bac e ial p eda o o ecognize, con ac , kill
and lyse he p ey, and use he by-p oduc s, bu also o he
down egula ed genes ha , in he case o he mul icellula
M. xan hus, a e especially impo an , because hey a e in cha ge
o di e ing he complex li ecycle owa ds p eda ion,
a oiding de elopmen .
He ein we ha e used RNA-seq echnology o de ine he
p eda osome o M. xan hus p eying on S. melilo i. The esul s
ob ained, in addi ion o iden i y which speci ic genes a e
up egula ed o de ec , kill, lyse, and consume he p ey, ha e
e ealed ha his myxobac e ium modi y i s lipid composi ion.
Mo eo e , compa ison wi h o he ansc ip omic changes du ing
p eda ion on o he p ey has allowed o ou line a co e p eda osome.
Finally, compa ison wi h he de elopmen al ansc ip ome has
allowed o d aw a global ansc ip ional pe spec i e o he
complex li ecycle o his in iguing mul icellula bac e ium.
Ma e ials and me hods
P epa a ion o M. xan hus synch onously
p eda o y cells
M. xan hus s ain DK1622 (Kaise , 1979; Goldman e al., 2006)
and S. melilo i Rm1021 (Meade and Signe , 1977) used in his s udy
we e g own in CTT (Hodgkin and Kaise , 1979) and TY (Be inge ,
1974) b o h, espec i ely, wi h igo ous shaking a 30°C. The
hizobial cul u e was g own o an op ical densi y a 600 nm (OD600)
o 1 and hen dilu ed o a inal OD
600
o 0.2. Fo each eplica e, wen 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 (1.5% Bac o-Aga [Di co] supplemen ed) and
incuba ed a 30°C o 24 h. Then, M. xan hus cells g own in CTT
liquid media o an OD600 o 1 we e cen i uged and concen a ed in
TM bu e (10 mM T is–HCl [pH 7.6], 1 mM MgSO4) o a inal
OD
600
o 15. Then, en-mic oli e s d ops o concen a ed cul u e o
he M. xan hus s ain we e deposi ed on op o each o he hizobial
colonies (samples Mx_Sm) o wen y 10-μl d ops we e placed on o
CTT pla es (samples Mx; Supplemen a y Figu e S1). A e 2 and 6 h
o incuba ion, wo 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 M. xan hus) we e
ha es ed om pla es, 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 M. xan hus = 0 samples
(samples Mx_ 0), wo eplica es o 3 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 250 μl 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 hylenediamine 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 Mini Ki (Qiagen),
pe o ming on-column DNase diges ion wi h he RNAse- ee
DNase se (Qiagen), and each sample was elu ed in 50 μl 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, Uni edS a es). To al RNA samples we e p ocessed
by No ogene (No ogene [Uni ed Kingdom] Company L d.) as
indica ed below.
Lib a y p epa a ion
To ob ain cDNA s and-speci ic lib a ies he NEBNex ®
Ul a™ Di ec ional RNA Lib a y P ep Ki (New England BioLabs,
Inc.) was used. B ie ly, a e passing an ini ial quali y con ol,
RNA was emo ed om o al RNA samples wi h he Illumina
Ribo-Ze o Plus RNA Reduc ion Ki (Illumina, Inc.), and
emaining RNA was andomly agmen ed. Fi s s and DNA was
syn he ized using andom hexame p ime s and in he nex s ep,
second-s and DNA was syn hesized. Double-s anded cDNA was
pu i ied using Agencou AMPu e XP Beads, and hen end- epai ,
polyadenyla ion and adap o -liga ion p ocesses we e pe o med
sequen ially. Nex , he liga ion eac ion was size-selec ed and
pu i ied using AMPu e XP Beads, and he p oduc s ob ained we e
used o PCR lib a y en ichmen . The PCR lib a y was pu i ied
using Agencou AMPu e XP Beads. Lib a ies we e quan i ied by
qPCR and inse size was de e mined using LabChip GX Nucleic
Acid Analyze (Pe kin-Elme Inc.).
Sequencing and global 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
No aSeq6000 (150 bp pai ed-end ead) sequencing pla o m
(No ogene [UK] Company L d.). 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 and/o S. melilo i Rm1021
genome sequences using Bow ie2 wi h he misma ch
pa ame e se o wo and o he pa ame e s se o de aul , using
he pai -end s a egy. Fo isualiza ion o mapping s a us o
eads iles we e p o ided in BAM o ma . A emis .18.0.0
(Ca e e al., 2012) was used o he isualiza ion o he
sequence eads agains he e e ence genomes. FPKM
( agmen s pe kilobase o ansc ip pe million agmen s
mapped) no maliza ion was used o compa ison o samples.
The a e age FPKM alues o he wo eplica es we e used o
calcula e he log2 old change.
On a e age, 16.16 million ead pai s and a co e age o 266X
we e ob ained. A e emo ing he ibosomal sequences, he
Pé ez e al. 10.3389/ micb.2022.1004476
F on ie s in Mic obiology 04 on ie sin.o g
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 DESeq2 R/EdgeR R package was used o iden i y
di e en ially exp essed ansc ip s (Robinson e al., 2010; Lo e
e al., 2014).
Resul s and discussion
Global da a o he ea ly ansc ip omic
esponse o M. xan hus on p eda o y
co-cul u es
When he p eda o con ac s wi h he p ey i mus quickly
and p ecisely adap o he new nu ien sou ce ha en ails a
li ing mic oo ganism. The esponse mus bespeci ic and will
e eal he mechanism used o p eda ion. He e, he ea ly
esponse o M. xan hus o he p ey S. melilo i has been analyzed
by collec ing co-cul u es samples a 2 and 6 h. We ha e
p e iously epo ed ha a la e imes mos p ey cells a e lysed
(Pé ez e al., 2014). A 2 h he p eda o has al eady es ablished
he i s con ac wi h he p ey and is ac i ely a acking.
Compa ison wi h a longe ime (6 h) will allow us o know
which genes a e impo an in he i s a ack and which a e
con inuously needed du ing p eda ion.
To examine ansc ip ional changes, RNA-seq echnology was
used. In his s udy, i e cDNA lib a ies we e cons uc ed: Mx_ 0:
M. xan hus alone a ime 0 h; Mx_ 2: M. xan hus alone collec ed
a e 2 h in solid CTT medium; Mx_ 6: M. xan hus alone g own
o 6 h in solid CTT medium; Mx_Sm 2: cells collec ed a e 2 h o
in e ac ion M. xan hus-S. melilo i; and Mx_Sm 6: whe e cells we e
ha es ed a e 6 h o in e ac ion M. xan hus-S. melilo i. Fo
simplici y, wewill e m 2 and 6 he 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 eda o cells in pu e cul u e a 2 o 6 h: Mx_ 2 and Mx_ 6,
espec i ely).
The o al aw eads gene a ed om each sample, he clean
eads ob ained a e emo al om he aw eads hose con aining
adap e s and/o low quali y, he e o s Q20 and Q30, and GC
con en o he clean eads a e summa ized in Supplemen a y
Table S1A.
Compa ison be ween he wo eplica es o each condi ion
showed a Pea son co ela ion coe icien (R2) close o 1 (all ≥0.947;
Supplemen a y Figu e S2A). The median o bo h alues was used
o u he analyses. To es ablish ela ionships be ween gene
exp ession p o iles o all he samples, wepe o med a p incipal
componen analysis (PCA) analysis. As expec ed, genes o he
same condi ion clus e oge he and genes o di e en nu i ional
s age and imes clus e sepa a ely (Supplemen a y Figu e S2B).
The mRNA mean eads we e no malized o FPKM alues
(Supplemen a y Table S1B). Compa isons o FPKM alues
be ween samples exhibi ed simila mRNA exp ession le els and
he iolin diag am o FPKM isually showed he gene exp ession
le els (Supplemen a y Figu es S2C,D). All hese da a show ha
he ansc ip ome is o good quali y.
In ou compa ison analysis wewill use he old nomencla u e
(MXAN_), since i is mos equen ly used in he li e a u e.
Howe e , in all he ables he co esponding new localize s
MXAN_RS a e also indica ed.
Func ional en ichmen and di e en ially
exp essed genes (DEGs) in co-cul u e
The ansc ip s o co-cul u es a 2 and 6 h e sus hei
espec i e con ols we e il e ed by hei old changes (|Log2 Fold
Change| > 0) and padj<0.05 o cons uc he olcano plo s o in e
he o e all dis ibu ion (Figu es1A,B). In o al, wede ec ed ha
277 a 2 h and 672 a 6 h ansc ip s we e up egula ed (including
sRNAs, i.e., non-coding RNA o 50–500 n leng h), while 194 a
2 h and 664 a 6 h we e down egula ed
(Supplemen a y Tables S1C,D).
To elucida e he biological p ocesses associa ed o DEGs
du ing p eda ion, 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 analysis o
up egula ed genes showed ha a 2 h (Mx_Sm 2 s. Mx_ 2) he
p o iles o signi ican ly en iched unc ions and pa hways change
d as ically compa ed o 6 h (Mx_Sm 6 s. Mx_ 6;
Supplemen a y Figu e S3A; Supplemen a y Tables S1E,F). A 2 h,
he p edominan pa hways a e in ol ed in a y acid (FA)
me abolism, seconda y me aboli e biosyn hesis, oxida i e
phospho yla ion, and wo-componen sys ems
(Supplemen a y Figu e S3A). Howe e , a 6 h, cells p io i ize
ansla ion, since many induced genes code o s uc u al
cons i uen o ibosomes, pep ide biosyn hesis, RNA, and p o ein
me abolism (Supplemen a y Figu e S3A).
Signi ican ly pa hways de ec ed among down egula ed genes a 2 and
6 h a e hose in ol ed in wo-componen sys ems and bac e ial chemo axis
(Supplemen a y Figu e S3B; Supplemen a y Tables S1G, H).
All hese DEGs a 2 and 6 h we e u he manually analyzed
and compa ed o he li e a u e (Supplemen a y Tables S2A,B).
Weha e excluded no el genes and sRNAs in hese s udies o
compa e ou da a wi h he ac ual knowledge abou he li ecycle o
M. xan hus. Weha e also conside ed in ou analyses genes ha a e
p edic ably encoded in he same clus e o in he same ope on
acco ding o he loca ions o eads in he e e ence genome,
ansc ip ion s a si es, and ansc ip ion e mina ion si es o
ope ons by using Rockhoppe sys em (Tjaden, 2020).
Fo a be e pic u e o he global changes ha ake place
du ing co-cul u e and o deciphe he p eda osome, weha e
conside ed all he up and down egula ed genes wi h |Log2
Fold Change|>0 and padj<0.05. The eason is ha wea e
compa ing p eda ion e sus g ow h on ich media and,
he e o e, any change in gene exp ession, no ma e how
small, mus be conside ed, because he di e ences mus
Pé ez e al. 10.3389/ micb.2022.1004476
F on ie s in Mic obiology 05 on ie sin.o g
bea ibu ed o he p esence o li ing p ey as nu ien s. I has
been also aken unde conside a ion in ou analysis hose
genes ha a e in ope ons o clus e s, o hose which unc ions
o implica ions in di e en me abolic pa hways ha e been
p e iously desc ibed. Fo imp o ed con idence, a h eshold o
Log2 Fold Change|>1 is indica ed in he all he igu es.
Howe e , i should be conside ed ha some de ensi e o
an agonis ic esponses o S. melilo i agains M. xan hus would
impac gene exp ession o he p eda o du ing he in e ac ion.
This in e ac i e esponse is unde esea ch in ou labo a o y.
Hyd oly ic enzymes and ex acellula p o eins
Fo y- ou genes coding o hyd oly ic enzymes we e
up egula ed wi h Log2 Fold Change om 0.78 o 3.4 (Figu e2A):
20 pep idases, 5 alpha-be a old hyd olases, 5 nucleases, 3 lipases,
3 lac amases, 3 amidases, 1 phosphoes e ase, 1 sul a ase, 1
hioes e ase, 1 polysaccha ide lyase, and 1 poly-hyd oxyalkanoa e
depolime ase. Mos o hese enzymes ha e ne e been s udied,
al hough some o hem ha e been associa ed o p eda ion. Thus,
he pep idase MepA has been sugges ed o beused as a seconda y
ac o du ing p eda ion, o b eak down p o eins eleased by
al eady lysed cells (Be leman e al., 2014); 3 o he pep idases ha e
been de ec ed in an ex acellula ac ion o M. xan hus ha
showed bac e ioly ic ac i i y (A end e al., 2021); and 5 o he
hyd oly ic enzymes ha e been expe imen ally loca ed in ou e
memb ane esicles (OMV): MXAN_0220, MXAN_2661 (Kahn
e al., 2010), MXAN_0976 (Zwa ycz e al., 2020), MXAN_0366,
and MepA (Be leman e al., 2014).
On he o he hand, o he 7 genes code o p o eins wi h no
hyd oly ic ac i i y, bu loca ed o en iched in OMVs, such as he
ou e memb ane p o ein Oa (Ma ínez-Cañame o e al., 1993),
which is a TonB-dependen anspo e in ol ed in sec e ion o
p o eins (Gómez-San os e  al., 2019). Since Oa is he majo
p o ein o he ou e memb ane o M. xan hus (Ma ínez-
Cañame o e al., 1993), i is plausible o specula e ha i may play
an essen ial ole in sec e ion o ac o s equi ed o p eda ion o
bac e ia, simila ly as i has been epo ed o he p eda ion o
ungi by myxobac e ia (Li e al., 2019).
Mo eo e , 5 genes encode p o eins holding pep idoglycan-
binding LysM domains (Figu e2B). These domains ha e been
ound in a wide a ie y o ex acellula p o eins and ecep o s
which a e esponsible o de ec ion o and binding o
pep idoglycans, and e en hey a e able o sense and disce n
be ween di e en mic oo ganisms (Wong e  al., 2019). Fo
ins ance, LysM o ms pa o he legume senso y sys em NFP
(Nod Fac o Pe cep ion) in ol ed in speci ically de ec i s
symbion bac e ia S. melilo i (Bensmihen e al., 2011). Simila ly, i
is expec ed ha hese M. xan hus p o eins may bein ol ed in
ecognizing speci ically he pep idoglycan o he p ey and i is no
uled ou ha hey a e able o disce n be ween
di e en mic oo ganisms.
These esul s show he ex ao dina y capaci y o M. xan hus
o ecognize, bind and lyse he p ey, and open he possibili y o
disco e new p oduc s.
S-mo ili y and Tad-like appa a us
Se e al genes coding o p o eins ela ed o M. xan hus
S-mo ili y a e sligh ly up egula ed a 6 h (Log2 Fold Change om
0.33 o 1.31), including he sigF sigma ac o , which is in ol ed in
he egula ion o genes o S-mo ili y (Ueki e al., 2005; Youde ian
and Ha zell, 2006). Se e al genes ound in his s udy a e in he
A
B
FIGURE1
Di e en ial exp ession esponse o M. xan hus du ing p eda ion.
Sc eening o di e en ially up egula ed (A) and down egula ed
(B) genes by olcano a 2 and 6in p eda o y condi ions.
Volcano plo s show he es ima ed old changes (x-axis) e sus
he minus log10 o he adjus ed p- alues (y-axis) om DEseq
analysis. Signi ican genes wi h absolu e alues o |Log2 Fold
Change|>0 and padj<0.05 a e shown in ed (up egula ed) o in
blue (down egula ed). Black e ical do ed line indica es ze o-
old change. G een do s indica e non- egula ed genes.

Pé ez e al. 10.3389/ micb.2022.1004476
F on ie s in Mic obiology 06 on ie sin.o g
same ope on (MXAN_0360–0363; F emgen e al., 2013) o in he
same clus e (pilA, pilO, pilS, pilQ; Wall and Kaise , 1999). On he
o he hand, 4 genes coding o he Tad-like appa a us (Kil p o eins)
a e up egula ed ei he a 2 o 6 h (Log2 Fold Change om 0.4 o
0.93): MXAN_3106 (kilC) and MXAN_4658–4660 (kilL, kilH and
kilM), which ha e been ecen ly desc ibed o be in ol ed in
con ac -dependen p ey killing (See e al., 2021; Figu e2C). These
esul s a e in good ag eemen wi h expe imen al da a ha ha e
demons a ed ha S-mo ili y (Pé ez e al., 2014) and he Tad-like
sys em (See e al., 2021) a e bo h impo an o p eda ion.
Seconda y me aboli es
M. xan hus p oduces a g ea a ie y o biologically ac i es
me aboli es. Howe e , only a ew o hem ha e been implica ed in
p eda ion. Mo eo e , many genes po en ially coding o enzymes
in ol ed in seconda y me aboli es biosyn hesis a e c yp ic in
labo a o y condi ions. In his analysis, i has been ound ha
du ing he in e ac ion wi h he p ey one clus e o genes
consis ing o 3 non- ibosomal pep ide syn he ases and 1 hyb id
non- ibosomal pep ide syn he ase/ ype I polyke ide syn hase
(MXAN_RS21375-MXAN_RS21390) is up egula ed (Log2 Fold
Change om 1.1 o 2.6) and, consequen ly, i mus bein ol ed in
he biosyn hesis o an uniden i ied bio-p oduc . A second clus e
(MXAN_3618–3620) is implica ed in he biosyn hesis o he
side opho e myxochelin (Ko p e  al., 2016, 2018; Figu e 2D;
Supplemen a y Table S2A). In addi ion, 5 ype I polyke ide
syn hases loca ed in he myxalamide clus e a e also up egula ed
a 6 h (MXAN_4526-MXAN_4530), sugges ing ha his an ibio ic
is also equi ed o killing he p ey, al hough u he esea ch is
equi ed. Simila ly, DK-xan hene also seems o play a ole in
p eda ion because 10 genes o he ope on a e also sligh ly
up egula ed (Figu e2D; Supplemen a y Table S2A). These esul s
ag ee wi h he inding ha DK-xan hene is o e p oduced du ing
p eda ion (Ellis e al., 2019). Finally, i is in e es ing o men ion
ha genes encoding enzymes in ol ed in he biosyn hesis o he
an ibio ics myxo i escin and myxop incomide a e no up egula ed
in he in e ac ion M. xan hus-S. melilo i, al hough hese wo
an ibio ics ha e been epo ed o bein ol ed in p eda ion o
M. xan hus on E. coli (Xiao e al., 2011) and B. sub ilis (Mülle
e al., 2016). The e o e, i emains o beelucida ed whe he hese
an ibio ics a e o e p oduced only on speci ic p ey, o whe he
hei o e p oduc ion is egula ed by a pos - ansc ip ional
mechanism. An in e es ing esul is he up egula ion (Log2 Fold
Change om 0.6 o 2.7) o genes in ol ed in geosmin biosyn hesis
(MXAN_6242–6257), including genes coding o a e pene
syn hase and wo ansc ip ional egula o s o he C p/Fn amily,
sugges ing ha his seconda y me aboli e somehow pa icipa es
in p eda ion (Figu e2D).
Ano he ema kable esul is he up egula ion (Log2 Fold
Change om 0.6 o 2.6) o a clus e (hslA, hslB, hslC, hslD, hslO,
hslP, hslQ), in ol ed in he syn hesis o homospe midine lipids
(Ho mann e al., 2018; Figu e2D). Homospe midine lipids a e
modi ied polyamines o med du ing M. xan hus de elopmen ,
which a e bioac i e agains a panel o mic oo ganisms (Ho mann
e  al., 2018). Ou da a indica e ha hey also pa icipa e in
p eda ion. Ho mann e al. (2018) p oposed ha hese seconda y
me aboli es a e o igina ed om a ginine, ia he pu escine
pa hway. The ac ha he comple e clus e (MXAN_5105–5110)
in ol ed in a ginine biosyn hesis is also up egula ed in p eda o y
condi ions ein o ces his sugges ion (Supplemen a y Table S2A).
These da a open he doo o iden i y new seconda y
me aboli es ha may ha e bio echnological applica ions.
Mo eo e , hey will help o ind new oles o seconda y
me aboli es ha ha e al eady been iden i ied.
Lipid me abolism
Many genes in ol ed in FA me abolism a e also up egula ed
du ing co-cul u e. I is no able o men ion s aigh -chain FA
biosyn hesis, since many genes in ol ed in hei syn hesis a e
up egula ed (Figu e 3; Supplemen a y Table S2A). Howe e ,
MXAN_0853, whose inac i a ion blocks comple ely he
p oduc ion in M. xan hus o s aigh -chain FA (Bode e al., 2006a),
is down egula ed a 2 h (Supplemen a y Table S2B). I could
bea gued ha gene MXAN_0215, which exhibi s high simila i y
o MXAN_0853 and is up egula ed, would ca y ou he same
unc ion (Figu e3), bu i has been epo ed ha i s dis up ion has
no e ec in lipid composi ion (Cu is, 2001; Bode e al., 2006a).
Consequen ly, MXAN_0215 migh ha e complemen a y
ABD
C
FIGURE2
Hea map wi h up egula ed genes du ing p eda ion a 2 and/o
6. (A) Hyd oly ic enzymes. (B) LysM p o eins. (C) S-mo ili y.
(D) Seconda y me aboli es. Red edges indica e genes wi h Log2
Fold Change > 1.
Pé ez e al. 10.3389/ micb.2022.1004476
F on ie s in Mic obiology 07 on ie sin.o g
unc ions wi h espec o s a ing uni speci ici y and migh
bein ol ed in he o ma ion o iso-FA du ing p eda ion.
Some o he up egula ed genes a e implica ed in he o ma ion
o iso ale yl-CoA (IV-CoA), which is he p ime o b anched-
chain iso-odd FA biosyn hesis. These FA a e esponsible o
main aining memb ane luidi y, and a e in ol ed in signaling
du ing de elopmen and in biosyn hesis o some seconda y
me aboli es (Bode e al., 2006b; Li e al., 2013). M. xan hus can
syn hesize IV-CoA ia leucine deg ada ion, bu also om
3-hyd oxy-me hyl-glu a yl-CoA (HMG_CoA) ia an al e na i e
sys em (aib ope on) om he me alona e pa hway (Figu e3; Bode
e al., 2009; Li e al., 2013; Oko h e al., 2020). As obse ed in
Figu e3, some genes o he Bkd/Esg complex (b anched-chain
α-ke o acid dehyd ogenase complex/E signal) in ol ed in IV-CoA
ia leucine deg ada ion a e down egula ed, bu genes om he
me alona e pa hway, including he aibC gene, which con e s
HMG-CoA in o IV-CoA, a e up egula ed. This al e na i e
pa hway is essen ial o ui ing body o ma ion, p esumably o
supply IV-CoA when leucine is limi ed (Bode e al., 2006b). Un il
now, he e a e no e idences ha his al e na i e pa hway unc ions
du ing g ow h. Howe e , ou esul s indica e ha his ou e is
ope a i e also in p eda o y condi ions (Figu e3). On he o he
hand, Pas e nak e al. (2013), compa ing he genomes o p eda o y
and non-p eda o y bac e ia, ound ha hey di e in isop enoid
biosyn hesis. While all p eda o s used he me alona e pa hway,
he non-me alona e pa hway is used by non-p eda o y bac e ia.
FIGURE3
Di e en ial exp ession du ing p eda ion a 2 and 6 o genes in ol ed in lipid biosyn hesis and deg ada ion, and hei possible implica ion in lipid
ex e nal use, and seconda y me aboli es biosyn hesis. Red ex indica es genes up egula ed du ing p eda ion. Blue ex indica es down egula ed
genes. In bold, |Log2 Fold Change|> 1.
Pé ez e al. 10.3389/ micb.2022.1004476
F on ie s in Mic obiology 08 on ie sin.o g
Ou esul s ein o ce he idea ha his me alona e ou e migh
beimpo an o bac e ial p eda ion. Fu he mo e, he me alona e
pa hway is also in ol ed in geosmin biosyn hesis in M. xan hus
(Bode e al., 2009; Ko p e al., 2016), indica ing again a ole o his
ola ile isop enoid in p eda ion.
Many genes coding o FA-elonga ion enzymes, ela ed o
sa u a ed FA biosyn hesis (Cu is and Shimke s, 2008), a e also
up egula ed in he p esence o he p ey, ein o cing he idea ha
lipid syn hesis is ac i a ed du ing p eda ion, mos likely o build
blocks o p eda o memb anes. Du ing g ow h M. xan hus
inco po a es p edominan ly unsa u a ed FA a sn-1 o he
phospholipids (Cu is e al., 2006). The sou ce o hese unsa u a ed
FA in M. xan hus is unknown, al hough desa u ases mus
bein ol ed. Howe e , du ing p eda ion, many genes ha code o
desa u ases a e signi ican ly down egula ed (Figu e 3;
Supplemen a y Table S2B), sugges ing ha M. xan hus is changing
he composi ion o he memb anes in he cell en elope in he
p esence o he p ey, inco po a ing mo e sa u a ed FAs. I is
emp ing o specula e ha hese changes may benecessa y in he
p eda o o de end om i s own a senal used du ing p eda ion.
Al hough es e bonds a e he mos common linkages in
phospha idyle hanolamine in Bac e ia, phospholipids con aining
e he -linked chains (e he lipids) a e p e alen in myxobac e ia
(Cu is and Shimke s, 2008). Mo eo e , only he b anched-chain
FA iso-de i a i es a e ound in e he -linked lipids (Ring e al.,
2009). These e he lipids, including plasmalogens, a e esponsible
o memb ane luidi y and con e s abili y o he cell agains
en i onmen al s esses (Cu is and Shimke s, 2008; Gallego-
Ga cía e al., 2019). Some genes coding o enzymes equi ed o
e he lipid syn hesis in M. xan hus, such as MXAN_1675 and
MXAN_1676, a e up egula ed du ing p eda ion. Howe e , since
hese genes also seem o bein ol ed also in he biosyn hesis o
homospe midine lipids, which ha e an ibac e ial p ope ies
(Ho mann e  al., 2018), hei ole in p eda ion emains o
beunco e ed.
Genes encoding enzymes in ol ed in β-oxida ion pa hways
a e also up egula ed, excep o he enzymes ha ca alyze he i s
s ep and he a y acyl-CoA ligases (FACL). FACLs ac i a e FA
be o e hey can beassimila ed in o a ious me abolic pa hways by
con e ing FA o FA-acyl-CoA. These bioac i e FA me aboli es, in
addi ion o se ing as subs a es o β-oxida ion and phospholipid
biosyn hesis, a e in ol ed in p o ein anspo , enzyme ac i a ion,
p o ein acyla ion, cell signaling, and ansc ip ional con ol
(Weima e al., 2002; DiRusso and Black, 2004). In M. xan hus
he e a e a leas 6 FACL pa alogues. Only one o hem,
MXAN_0216, is up egula ed, while ou o hem a e
down egula ed and he one emaining does no change i s
exp ession le els (Supplemen a y Figu e S4A). FACLs appea o
beme abolic signals o FA deg ada ion by bac e ia in gene al
(Weima e al., 2002), and he ac ha MXAN_0216 exp ession
is only de ec ed du ing p eda ion sugges s ha his FACL migh
ha e a unc ional ole in he ansmemb ane mo emen and
ac i a ion o exogenous speci ic FA be o e consuming hem as
ca bon o ene gy sou ces. Acco ding o he idea ha M. xan hus
uses p ey lipids as nu ien s is he up egula ion o genes in ol ed
in lipid deg ada ion, such as hose coding o lipases o es e ases
(Figu e 3). Howe e , he classical long-chain FA anspo e s
(FadL-like) a e down egula ed (MXAN_7040 and MXAN_6116)
and, consequen ly, he e mus unc ion in ano he unknown
up ake p ocess.
These da a indica e ha ce ain pa hways in ol ed in lipid
me abolism a e up egula ed du ing co-cul u e. I is expec ed ha
anabolic pa hways will p o ide he p eda o wi h ene gy. In
con as , anabolic pa hways seem o beused 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 he p ey.
I on up ake
Genes in ol ed in syn hesis and sec e ion o he high-
a ini y i on-chela ing side opho es, myxochelin A and
myxochelin B (Silakowski e al., 2000; Li e al., 2008), a TonB-
dependen anspo e (FepA-like; MXAN_6911) ha
ecognizes and impo s e ic side opho es, and a Fe-ABC
pe iplasmic i on compound binding p o ein anspo e
(MXAN_0684–0687) a e all up egula ed by M. xan hus when
p ey on S. melilo i. Mo eo e , he side opho e educ ase
(MXAN_3639) ha educes Fe
3+
o Fe
2+
in he cy oplasm is also
up egula ed (Figu e4; Supplemen a y Table S2A). These da a
indica e ha M. xan hus needs a high i on concen a ion and,
consequen ly, up egula es he i on up ake machine y p obably
o compe e o he me al in he p esence o p ey. The need o
i on o M. xan hus du ing co-cul u e is ein o ced by he
up egula ion o genes coding o heme-ca ie p o eins
implica ed in acquisi ion o i on om o he o ganisms’ i on-
binding p o eins (MXAN_1314–1321). The TonB complexes,
ha ene gize he ou e memb ane ecep o s, a e mos likely
MXAN_0819–0821 o MXAN_0273–0276 since some o hese
genes a e uniquely exp essed in p eda o y condi ions. O he
i on- ela ed genes coding o p o eins ha ha e been p e iously
de ec ed in a p o eome in i on-poo condi ions o ha bind o
he p edic ed global egula o y ep esso Fu (MXAN_3702) by
pull-down echnique (Al meye , 2010), a e also up egula ed
(Supplemen a y Table S2A). In addi ion, ou sea ch o Fu
boxes in M. xan hus genome using he Vi ual Foo p in
da abase (Münch e  al., 2005) agains he Fu ma ix o
Esche ichia coli, Helicobac e pylo i, Klebsiella pneumoniae,
Vib io chole a and Pseudomonas ae uginosa, plus a manual
sea ch, has de ec ed eigh een up egula ed genes wi h pu a i e
Fu p omo e s (Figu e4; Supplemen a y Table S2A). Maybe his
i on necessi y is o supply his me al as a co ac o o enzymes
equi ed o p ope p eda ion. In ac , a leas 14 genes ha code
o i on–sul u p o eins a e up egula ed du ing he p eda o y
p ocess. Al e na i ely, i on migh beused by M. xan hus as a
weapon o killing du ing p eda ion, as i has been epo ed
wi h coppe (Con e as-Mo eno e al., 2020). Al hough i on has
been ela ed wi h phase a ia ion in M. xan hus (Dziewanowska
e al., 2014), weha e no obse ed any changes du ing he
in e ac ion o he myxobac e ium on S. melilo i.
Pé ez e al. 10.3389/ micb.2022.1004476
F on ie s in Mic obiology 09 on ie sin.o g
Ribosomal biogenesis and ansla ion
Acco ding o he en ichmen analysis, a 6 h many genes
coding o p o eins in ol ed in ansla ion a e up egula ed du ing
co-cul u e, including genes ha code o ibosomal p o eins (17
genes o 30S p o eins and 27 genes o 50S p o eins;
Supplemen a y Figu e S4B), elonga ion ac o s (8), RNA ligases
(15), and e mina ion ac o s (2) (Supplemen a y Table S2A). This
esul indica es ha cells a 6 h a e s a ing o ac i ely syn he ize
p o eins. A his ime, p eda o cells ha e mo e nu ien s a ailable,
and i is expec ed ha hey inc ease hei g ow h a e. In addi ion,
hey mus con inue syn hesizing p o eins and seconda y
me aboli es o lyse he p ey.
T ansc ip ional egula o s
I has been obse ed ha only 24 genes coding o
ansc ip ional ac o s a e up egula ed du ing co-cul u e
(Supplemen a y Figu e S5A; Supplemen a y Table S2A). These
egula o y elemen s a e expec ed o modula e he exp ession o
genes ha acili a e killing and lysing o he p ey. In con as , he
numbe o genes coding o egula o y p o eins ha a e
down egula ed ises o 97 (Supplemen a y Figu e S5A;
Supplemen a y Table S2B). In e es ingly, many o hese egula o s
ha e been p e iously implica ed in he de elopmen al cycle
(Supplemen a y Figu e S5A; Supplemen a y Table S2B). The
ele ance o hese changes in egula ion will bediscussed below.
O he up egula ed genes
In addi ion o he genes and unc ions epo ed abo e, i is
no ewo hy o men ion o he genes ha seem o be ele an du ing
he p eda o y p ocess. Fo ins ance, 15 genes encode p o eins ha
o m pa o ABC anspo e s (eigh o hem in ol ed in i on
up ake). Fu he mo e, 3 genes belong o he Majo Facili a o s
Supe amily (MFS), p obably in ol ed in he expo o hyd oly ic
enzymes o seconda y me aboli es. Finally, wo clus e s ela ed o
cy och ome c oxidase cbb3- ype biogenesis, membe s o he
heme-Cu oxidase supe amily (MXAN_5538–5541 and
MXAN_5553–5557), a e up egula ed wi h he highes Log2 Fold
Change ob ained in his ansc ip ome (Supplemen a y Table S2A).
Since hose e minal oxidases a e speci ically equi ed a low
oxygen ensions (Pi che and Wa mough, 2004), i is emp ing o
specula e ha he con ac wi h he p ey c ea es a mic oae ophilic
a mosphe e ha would equi e o p o eins ha wo k mo e
e icien ly in hese condi ions.
Sea ching o he co e M. xan hus
p eda osome
The in e ac ion M. xan hus-p ey seems o di e om one p ey
o ano he , al hough he p ey ange canno bedi ec ly co ela ed
wi h he p ey phylogeny (Li ings one e al., 2017; A end e al.,
2021). Fo ins ance, he s a egies used by M. xan hus o
p eda ion on se e al s ains o S. melilo i a e di e en depending
on he galac oglucan p oduc ion (Pé ez e al., 2014). Howe e ,
he e a e also some common ea u es, like he ecen ly desc ibed
Tad-like machine y ha seems o beessen ial o p eda ion on
di e en p eys (See e al., 2021).
M. xan hus p eda osomes a ailable so a ha e been ob ained
no only using di e en p ey, bu also unde di e en labo a o y
condi ions, so i is di icul o d aw conclusions abou common
ansc ip ional changes ha may occu when he p eda o
con on s he p ey. Some ansc ip omic changes migh bedue o
di e si y o condi ions, such as nu ien s in he cul u e media
A
B
C
FIGURE4
I on up ake mechanisms a e induced in co-cul u e. (A) Genes
p edic ed o bein ol ed in i on up ake and ha a e up egula ed
in co-cul u e a 2 and/o 6. The numbe s o he genes depic ed
in he igu e a e he co esponding MXAN_ iden i ie s.
(B) G aphical ep esen a ion o he clus e o genes in ol ed in
myxochelin biosyn hesis. The se en up egula ed ansc ip s a e
indica ed by hei co esponding gene name. (C) Fu box (FB)
p edic ed by using he Vi ual Foo p in da abase. Red balls: Fe3+;
blue balls: Fe2+. OM, ou e memb ane; IM, inne memb ane. Fo
mo e de ails please see he ex and Supplemen a y Table S2A.