Ci a ion: Ma cos-To es, F.J.;
Mo aleda-Muñoz, A.;
Con e as-Mo eno, F.J.;
Muñoz-Do ado, J.; Pé ez, J.
Mechanisms o Ac ion o
Non-Canonical ECF Sigma Fac o s.
In . J. Mol. Sci. 2022,23, 3601.
h ps://doi.o g/10.3390/
ijms23073601
Academic Edi o : Jan Ko manec
Recei ed: 7 Ma ch 2022
Accep ed: 24 Ma ch 2022
Published: 25 Ma ch 2022
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In e na ional Jou nal o
Molecula Sciences
Re iew
Mechanisms o Ac ion o Non-Canonical ECF Sigma Fac o s
F ancisco Ja ie Ma cos-To es 1, Au elio Mo aleda-Muñoz 2,* , F ancisco Ja ie Con e as-Mo eno 2,
JoséMuñoz-Do ado 2and Juana Pé ez 2,*
1Es ación Expe imen al del Zaidín, Consejo Supe io de In es igaciones Cien í icas, 18011 G anada, Spain;
jma cos@ug .es
2Depa amen o de Mic obiología, Facul ad de Ciencias, Uni e sidad de G anada, A da, Fuen enue a s/n,
18071 G anada, Spain; cu ocm@ug .es (F.J.C.-M.); jdo ado@ug .es (J.M.-D.)
*Co espondence: au eliom@ug .es (A.M.-M.); jp o es@ug .es (J.P.); Tel.: +34-95-824-2858 (A.M.-M.);
+34-95-824-9830 (J.P.)
Abs ac :
Ex acy oplasmic unc ion (ECF) sigma ac o s a e subuni s o he RNA polyme ase spe-
cialized in ac i a ing he ansc ip ion o a subse o genes esponding o a speci ic en i onmen al
condi ion. The signal- ansduc ion pa hways whe e hey pa icipa e can be ac i a ed by di e se
mechanisms. The mos common mechanism in ol es he ac ion o a memb ane-bound an i-sigma
ac o , which seques e s he ECF sigma ac o , and eleases i a e he s imulus is sensed. Howe e ,
despi e mos o hese sys ems ollowing his canonical egula ion, he e a e many ECF sigma ac o s
exhibi ing a non-canonical egula o y mechanism. In his e iew, we aim o p o ide an upda ed and
comp ehensi e iew o he di e en ac i a ion mechanisms known o non-canonical ECF sigma ac-
o s, de ailing hei inclusion o he di e en phylogene ic g oups and desc ibing he mechanisms o
egula ion o some o hei ep esen a i e membe s such as Ec G om Rhodobac e sphae oides, showing
a pa ne -swi ch mechanism; Ec P om Vib io pa ahaemoly icus, wi h a phospho yla ion-dependen
mechanism; o Co E om Myxococcus xan hus, egula ed by a me al-sensing C- e minal ex ension.
Keywo ds: ECF sigma ac o ; ansc ip ional egula ion; s ess esponse
1. In oduc ion
In o de o su i e o he e e -changing en i onmen al condi ions, bac e ia ha e
de eloped a se o di e se egula o y mechanisms o sense and espond o ex e nal and
in e nal signals. These signal- ansduc ion mechanisms a e gene ally classi ied in ou main
g oups, also known as he ou pilla s o signal ansduc ion: one- and wo-componen
sys ems, ex acy oplasmic unc ion (ECF) sigma ac o s, and Se /Th p o ein kinases
(STPK) [
1
,
2
]. Sigma ac o s a e he subuni s o he RNA polyme ase (RNAP) in ol ed in
p omo e ecogni ion and ini ia ion o he ansc ip ion p ocess [
3
,
4
]. ECF sigma ac o s
ep esen a speci ic g oup o hese subuni s (G oup 4 o sigma ac o s), ha bo ing only wo
o he ou conse ed domains o sigma ac o s (σ2 and σ4) [3].
ECF sigma ac o s a e specialized in he esponse o speci ic condi ions, such as
en i onmen al s ess, di e en ia ion, o li e cycle s age. Typically, in he absence o he
s imulus, ECF sigma ac o s a e kep seques e ed by hei co- ansc ibed an i-sigma ac o s.
Upon he a i al o he s imulus, hese memb ane-ancho ed an i-sigma ac o s ac as he
senso pa o hese signal- ansduc ion mechanisms and elease he sigma ac o , no mally
h ough he egula ed in amemb ane p o eolysis (RIP) o he an i-sigma ac o , o s a
he ansc ip ion o genes in esponse o he igge ing s imulus. E en hough his would
ep esen he mos common and widesp ead mode o egula ion o canonical ECF sigma
ac o s, a b oad di e si y o egula o y mechanisms has been desc ibed o hese p o eins
since hei disco e y [
5
–
7
]. This di e si y was i s e iden a e he phylogene ic analysis
pe o med by S a o´n e al. in 2009, which esul ed in he es ablishmen o 66 ECF sigma
ac o g oups wi h e y dis inc i e ea u es [
1
]. This classi ica ion, plus he la e addi ions
In . J. Mol. Sci. 2022,23, 3601. h ps://doi.o g/10.3390/ijms23073601 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2022,23, 3601 2 o 13
summing up o mo e han 94 g oups, was used o de ine he di e en mechanisms o
ac i a ion o ECF sigma ac o s [
5
,
6
]. Howe e , since he las a emp o do such a egula o y
classi ica ion by Pin o and Masche in 2016, new disco e ies ega ding ECF sigma ac o s
ha e been published, including he mos ecen phylogene ic analysis pe o med by Casas-
Pas o e al. in 2021, which esul ed in he o ma ion o 157 new ECF g oups, wi h he
e-de ini ion, disappea ance, o expansion o many o he o iginal g oups [
6
,
7
]. Many
de ined ECF g oups con ain only a single sigma membe . This makes he sigma g ouping
p oblema ic and shows a wide a ie y o sigma ypes. In his mini- e iew, we aim o
p o ide an upda ed gene al and comp ehensi e iew o he complexi y o egula o y
mechanisms o he non-canonical ECF sigma ac o s.
Wha we de ine he e as canonical ECF sigma ac o s a e hose ollowing he gene al
mechanism o egula ion o he majo i y o ECF sigma ac o s. These canonical egula o s
a e unde con ol o hei cogna e memb ane-bound an i-sigma ac o s, ans e ing an
ex acy oplasmic signal ac oss he bac e ial memb anes o igge a gene ic esponse,
ega dless o he use o addi ional egula o y elemen s as in he case o cell-su ace signaling
sys ems o he an i-an i-sigma ac o s. Thus, he non-canonical ECF sigma ac o s would
be hose whose egula o y mechanisms in ol ing a cy oplasmic signal, o whe e he signal
ansduc ion ac oss memb anes in ol es he ac ion o addi ional mechanisms, such as wo-
componen sys ems. Acco ding o his de ini ion, we can dis inguish be ween wo g oups
o non-canonical ECF sigma ac o s: (1) hose unde he con ol o a soluble an i-sigma
ac o , and (2) hose no associa ed o an an i-sigma ac o .
2. ECF Sigma Fac o s wi h a Soluble An i-Sigma Fac o
E en hough egula o y soluble an i-sigma ac o s no mally sha e li le sequence
homology wi h hei memb ane-bound unc ional homologues, hey do sha e a s iking
s uc u al homology leading o he cha ac e iza ion o he an i-sigma domain p esen in
mos o hese p o eins [
8
]. This di e si y o an i-sigma ac o s makes he iden i ica ion
o some o hei mos di e gen membe s a complica ed ask, esul ing in many p o eins
ha ing a pu a i e an i-sigma ac o s a us.
Th ee mechanisms o egula ion ha e been epo ed o ECF sigma ac o s ha unc ion
wi h a soluble an i-sigma ac o (Table 1): sigma ac o s egula ed by con o ma ional
change, sigma ac o s egula ed by pa ne swi ch, and sigma ac o s egula ed by a
mechanosensing complex.
Table 1.
Di e en egula o y mechanisms o he ECF sigma ac o s wi h soluble an i-sigma ac o s.
The ECF g oups a e hose ha appea in he new classi ica ion o Casas-Pas o e al., 2021 [
7
]. * Only
ce ain membe s o he g oup.
P oposed Mechanism ECF G oup Model Sigma Fac o Re e ence
ECF11 RpoE om Rhodobac e sphae oides [9]
ECF12 SigH om Mycobac e ium ube culosis [10]
ECF14 SigE om Mycobac e ium ube culosis [11]
ECF19 * WP_016472479.1 om S ep omyces albus [7]
Con o ma ional change
ECF293 * RpoE om Neisse ia meningi idis [12]
Pa ne swi ch ECF15 Ec G om Me hylobac e ium ex o quens [13]
Mechanosensing ECF102 SigX om Pseudomonas ae uginosa [14]
ECF125 WP_044516075.1 om
Mycolicibac e ium sep icum [7]
ECF127 EJO88542.1 om Mycobac e ium colombiense [7]
ECF270 ODS58609.1 om Acidobac e ia bac e ium
SCN 69–37 [7]
ECF271 OGO36537.1 om Chlo o lexi bac e ium
RBG_16_56_8 [7]
ECF286 WP_003983642.1 om S ep omyces imosus [7]
Unknown mechanism
ECF292 WP_036395736.1 om
Mycolicibac e ium cosme icum [7]
O he less-unde s ood ECF sigma ac o s wi h soluble an i-sigma ac o s encompass
hose included in g oups ECF125 and ECF127, which a e encoded nex o a gene wi h
In . J. Mol. Sci. 2022,23, 3601 3 o 13
high simila i y wi h he an i-sigma ac o s o g oup ECF121; hose included in g oups
ECF270 and ECF271, which a e egula ed by p o eins wi h a Zn-dependen an i-sigma
domain (ZAS); and hose included in g oups ECF286 and ECF292, which a e encoded
nex o soluble p o eins om he Asp23 amily, p edic ed o ac as an i-sigma ac o s [
7
].
Howe e , due o he lack o expe imen al da a o con i m hei ac i a ion mechanisms,
hese ECF sigma ac o s will no be discussed in his e iew.
2.1. ECF Sigma Fac o s Regula ed by Con o ma ional Change
These ECF sigma ac o s ep esen he closes ac i a ion mechanism o he canonical
one. Howe e , con a y o he memb ane-bound an i-sigma ac o s, whe e he loca ion o
he senso domain enables hem o espond o signals p esen in he pe iplasm o he ou e
memb ane, hese soluble p o eins gene ally espond o cy oplasmic signals. These soluble
an i-sigma ac o s b andish a ZAS domain, which no mally esponds o di e en ypes
o oxida i e s ess. Examples o his mechanism would include RpoE om Rhodobac e
sphae oides (ECF11), and SigH and SigE om Mycobac e ium ube culosis (ECF12 and ECF14,
espec i ely) [9–11].
RpoE is he mas e egula o o he single oxygen and o ganope oxides oxida i e
s ess esponse in R. sphae oides. Unde no mal condi ions, RpoE is kep inac i e by he
soluble p o ein Ch R, which in e ac s wi h he egions binding o he RNAP and he DNA,
ac ing as an an i-sigma ac o . Ch R egula o s ha e wo well-de ined domains: an N-
e minal ZAS domain, and a C- e minal cupin-like domain (CLD). Whe eas he i s one
is cha ac e ized by he wo conse ed cys eines ha coo dina e he binding o Zn
2+
o
seques e he ECF sigma ac o , he las one would ac as he senso egion o his p o ein,
also binding o a Zn
2+
a om [
9
]. E en hough in many oxida i e s ess sensing ZAS p o eins,
he dissocia ion be ween sigma and an i-sigma p o eins akes place by he con o ma ional
change de i ed om he oxida i e damage o he Zn
2+
ligands [
15
], i has been shown
ha single oxygen s imula es Ch R p o eolysis [
16
]. Howe e , i is unclea whe he his
u no e plays a key ole in he elease o RpoE o how his egula ed p o eolysis may
occu [9,17].
The oxida i e-s ess sigma ac o s SigH and SigE om M. ube culosis a e egula ed by
he soluble p o eins RshA and RseA, espec i ely (Figu e 1A). These egula o y p o eins
ha e a ZAS domain ha , as men ioned abo e, coo dina es a Zn
2+
a om o bind o hei
espec i e ECF sigma ac o s and keep hem inac i e. Upon oxida i e s ess condi ions,
he Zn
2+
ligand is eleased om bo h p o eins, causing a con o ma ional change ha
dis up s hei binding o hei ECF sigma ac o s [
10
,
11
,
18
]. Bo h ECF sigma ac o s ha e
an addi ional le el o egula ion media ed by he Se /Th p o ein kinase PknB, since hese
sigma ac o s a e also eleased upon phospho yla ion o he an i-sigma ac o [18–20].
Figu e 1.
Regula ion o non-canonical ECF sigma ac o s associa ed o soluble an i-sigma ac o s,
exempli ied by known ECFs and ac i a ing s esses. ECF sigma ac o s a e depic ed in blue, whe eas
p o eins ac ing as an i-sigma ac o s and o he egula o y p o eins a e depic ed in o ange and pink,
espec i ely. (
A
) Regula ion by con o ma ional change: RshA and SigH om M. ube culosis; (
B
) Pa -
ne swi ch in NepR-PhyR and Ec G complex om R. sphae oides; (C) Model o he mechanosensing
complex go e ning he esponse o SigX in P. ae uginosa. OM: ou e memb ane; CM: cy oplas-
mic memb ane.
In . J. Mol. Sci. 2022,23, 3601 4 o 13
2.2. ECF Sigma Fac o s Regula ed by Pa ne Swi ch
ECF sigma ac o s egula ed by pa ne swi ch, also known as sigma ac o mimic y,
a e no mally kep inac i e by a soluble p o ein ac ing as an an i-sigma ac o . Howe e , in
his case, he signaling mechanism addi ionally in ol es he ac ion o a wo-componen
sys em, consis ing o a his idine kinase and a esponse egula o . Con a y o o he sys ems,
he e he an i-sigma ac o does no ha e a senso y ole, bu simply p e en s he ECF sigma
ac o om binding o hei p omo e sequences. The s imulus is ins ead de ec ed by he
his idine kinase which, in u n, will phospho yla e i s cogna e esponse egula o . The
esponse egula o has a egion wi h a high s uc u al simila i y o he ECF sigma ac o
i sel and, once phospho yla ed, binds o he an i-sigma ac o mo e e icien ly han o he
ECF sigma ac o . The bes unde s ood ECF sigma ac o egula ed by pa ne swi ch is
Ec G om Me hylobac e ium ex o quens (ECF15). Ec G is he cen al egula o o he gene al
s ess esponse in Alphap o eobac e ia [
13
]. In he absence o s ess, he an i-sigma ac o
NepR binds o he sigma ac o Ec G and keeps i inac i e. Upon s ess condi ions, he
esponse egula o PhyR ge s phospho yla ed by i s cogna e his idine kinase and mimics
he sigma ac o Ec G, eplacing i on he NepR binding si e, and eleasing he ac i e Ec G
(Figu e 1B). This mechanism, a he han as a pai ed sys em, is p oposed o wo k as a
e na y complex, whe e NepR would be equi ed o PhyR co ec phospho yla ion and
subsequen con o ma ional change [
13
]. This complex o ma ion seems o be media ed
by he in insically diso de ed egion o he an i-sigma ac o NepR, which may become
s uc u ed o in e ac wi h PhyR [21].
2.3. ECF Sigma Fac o s Regula ed by a Mechanosensing Complex
This mechanism o egula ion is one o he la es addi ions o his classi ica ion, and
coun s wi h only one s udied case: SigX om Pseudomonas ae uginosa (ECF102). The
pu a i e an i-sigma ac o is a soluble small p o ein named C X. This p o ein has been
p oposed o be pa o a mechanosensing complex oge he wi h he ou e memb ane po in
Op F, and he ion channel CmpX ha coo dina es ac i a ion o SigX (Figu e 1C). SigX is
in ol ed in he esponse o memb ane s ess and cold-shock, and egula es a ple ho a o
genes in ol ed in mo ili y, i on up ake, cell wall in eg i y, a y acids biosyn hesis, and
i ulence [14,22–24].
3. ECF Sigma Fac o s No Associa ed wi h an An i-Sigma Fac o
This g oup in ol es he mos di e se and pe haps he mos in e es ing ECF sigma
ac o s om an e olu iona y and bio echnological poin o iew. The independence o
many o hese egula o s om addi ional p o eins bypasses he limi a ions o hei use o
he e ologous exp ession and, in some cases, can e lec many o he gene usion e en s
happening h oughou bac e ial e olu ion [
25
]. The many egula o y mechanisms known
o hese ECF sigma ac o s include ansc ip ional egula ion, con o ma ional changes,
p o eolysis, phospho yla ion, and he use o N- and C- e minal egula o y ex ensions
(Table 2).
Besides he al eady well-de ined egula o y mechanisms, he e a e many g oups o
ECF sigma ac o s wi hou cogna e an i-sigma ac o s ha a e po en ially egula ed by a
non-canonical mechanism ye o be cha ac e ized. Among he conse ed p o eins in hei
gene ic neighbo hood ha could be in ol ed in hei egula o y mechanisms a e ound
p o eins wi h a DUF3470 and an i on-sul u binding domain (ECF58), 6-O-me hylguanine
DNA me hyl ans e ases (ECF122), glycosyl ans e ases (ECF248), ABC anspo e s and
AAA ATPases (ECF257), and PadR ansc ip ional ep esso s (ECF265) [7].
In . J. Mol. Sci. 2022,23, 3601 5 o 13
Table 2.
Regula o y mechanisms o he ECF sigma ac o s wi hou an i-sigma ac o . The ECF g oups
a e hose ha appea in he new classi ica ion by Casas-Pas o e al., 2021 [
7
]. * Only ce ain membe s
o he g oup.
P oposed Mechanism ECF G oup Model Sigma Fac o Re e ences
ECF12 * ECF12s9 and ECF12s2 om Mycobac e ium sp. [7]
ECF32 H pL om Pseudomonas sy ingae [26]
ECF39 * SigE om S ep omyces coelicolo [27]
ECF114 SigH om Po phy omonas gingi alis [28]
ECF203 SCD72908.1 om S ep omyces sp. D alAA-19 [7]
ECF234 APQ59451.1 om Paenibacillus polymyxa [7]
T ansc ip ional egula ion
ECF293 * PA3285 om Pseudomonas ae uginosa [14]
Con o ma ional changes ECF36 * SigC om Mycobac e ium ube culosis [7]
ECF54 SFT86700.1 om Geode ma ophilus ama gosae [7]
P o eolysis ECF282 An A om S ep omyces albus [29]
ECF43 Ec P om Vib io pa ahaemoly icus [30]
ECF59 SFI47409.1 om Planc omic obium pi i o me [7]
ECF61 OJW24604.1 om Planc omyce ales bac e ium
71–10 [7]
ECF62 WP_008685225.1 om Rhodopi ellula sallen ina [7]
ECF217 ELP31162.1 om Rhodopi ellula bal ica [7]
Phospho yla ion
ECF283 WP_056749340.1 om Noca dioides sp. Roo 190 [7]
ECF41 SigJ om Mycobac e ium ube culosis [31]
Con o ma ional change ECF238 Co E om Myxococcus xan hus [32,33]
ECF42 S en_0747 om S ep omyces enezuelae [31]
P o ein in e ac ion ECF57 * WP_015250107.1 om Singulisphae a acidiphila [6]
ECF36 * KLO31890.1 om Mycolicibac e he aklionensis [7]
ECF48 WP_048473130.1 om Mycolicibac e ium
chlo ophenolicum [7]
ECF52 SCO4117 om S ep omyces coelicolo [34]
ECF53 WP_030276194.1 om S ep omyces
pu peoch omogenes [7]
ECF115 KOP67510.1 om Bacillus sp. FJAT-18019 [7]
ECF243 * Iu Y om Pseudomonas pu ida [35]
P o eolysis
ECF270 * WP_011419852.1 om Anae omyxobac e
dehalogenans [7]
ECF29 SED43577.1 om B ady hizobium lablabi [7]
ECF56 WP_042440600.1 om S ep acidiphilus albus [7]
ECF123 *
WP_028426757.1 om S ep omyces sp. TAA040
[7]
ECF205
WP_019068201.1 om S ep omyces hoku onensis
[7]
ECF216 * QDE78790.1 om Myxococcus xan hus [7]
ECF220 WP_061622786.1 om So angium cellulosum [7]
ECF237 OLT65459.1 om Moo ea p oducens [7]
ECF240 *
SIO28919.1 om Ch yseobac e ium scoph halmum
[7]
ECF262 SFB89493.1 om Ruminococcus albus [7]
ECF264 WP_037286607.1 om Saccha ibacillus saccha i [7]
ECF276 * WP_063815919.1 om So angium cellulosum [7]
ECF287 WP_033089221.1 om Noca dia se iolae [7]
ECF288 WP_018594055.1 om Blau ia p oduc a [7]
ECF294 AKZ62584.1 om He baspi illum hil ne i [7]
Wi h egula o y ex ensions
O he s
ECF295 WP_063065904.1 Noca dia iolaceo usca [7]
ECF58 APZ92118.1 om Fue s ia ma isge manicae [7]
ECF122
WP_057211282.1 om Cellulomonas sp. Roo 930
[7]
ECF201 CDO03659.1 om Oceanobacillus pic u ae [7]
ECF248 EOZ99538.1 om Indibac e alkaliphilus [7]
ECF257 WP_010287217.1 om Ku hia massiliensis [7]
Unknown mechanism
ECF265 * AKO94994.1 om Bacillus endophy icus [7]
3.1. ECF Sigma Fac o s T ansc ip ionally Regula ed
These ECF sigma ac o s a e exp essed unde con ol o ano he signal- ansduc ion
mechanism, which di ec ly ac i a es he ansc ip ion o he ac i e ECF sigma ac o in a
cascade ashion.
One o he bes known ECF sigma ac o s egula ed by his mechanism is H pL om
Pseudomonas sy ingae (ECF32). The exp ession o his egula o is unde con ol o he
enhance -binding p o ein complex H pRS, which ac i a es ansc ip ion o he h pL gene
om a sigma-54 dependen p omo e [
36
]. H pL is in ol ed in he egula ion o mos ype
In . J. Mol. Sci. 2022,23, 3601 6 o 13
3 sec e ion sys em (T3SS) genes, which a e equi ed o plan in ec ion by his pa hogen.
The egula o y cascade con olling exp ession o h pL can expand beyond H pRS, in ol ing
mo e han 20 ac o s—such as he wo-componen sys ems RhpRS and Co RS, he one-
componen sys em Ae R, o he p o ease LonD—and can espond o en i onmen al cues
such as changes in nu ien le els, empe a u e, o osmo ic p essu e [
26
,
36
]. The main
nega i e egula ion o his mechanism is pe o med by H pL i sel in a nega i e eedback
ashion, whe e he complex o med by he RNAP wi h H pL blocks he ansc ip ion om
he sigma-54 dependen p omo e [37].
O he well-known examples would include SigE and SigQ om S ep omyces coelicolo
(ECF39). SigE is unde con ol o he wo-componen sys em CseBC and he lipop o ein
CseA, which espond o cell-wall damage caused by a di e se se o s ess ac o s such
as lysozyme o an ibio ics a ge ing he pep idoglycan, such as ampicillin o ancomycin
(Figu e 2A) [
27
,
38
]. SigQ, on he o he hand, is unde con ol o he wo-componen sys em
A sQ1/Q2, and egula es spo ula ion and he syn hesis o many an ibio ics, p obably in
esponse o al e a ions in he ni ogen me abolism o he C/N/P a io [39,40].
Figu e 2.
Examples o desc ibed egula o y mechanisms in ECF sigma ac o s non-associa ed o an
an i-sigma ac o . ECF sigma ac o s a e depic ed in blue, whe eas addi ional egula o y p o eins
a e depic ed in pink. (
A
) SigE om S. coelicolo is ansc ip ionally egula ed by he wo-componen
sys em CseBC, which esponds o cell wall damage; (
B
) SigC, in ol ed in i ulence o M. ube culosis,
is egula ed by a con o ma ional change unde in ec ion condi ions; (
C
) An A om S. albus is
deg aded by ClpXP du ing ce ain s ages o he li e cycle; (
D
) Ec P om V. pa ahaemoly icus is
ac i a ed by phospho yla ion by he STPK PknT in esponse o he an ibio ic polymyxin. OM: ou e
memb ane; CM: cy oplasmic memb ane.
Besides hese known examples, se e al ECF sigma ac o s ha e been p oposed o be
ansc ip ionally egula ed. Thus, he sigma ac o PA3285 (ECF293) om P. ae uginosa is
p edic ed o be egula ed by he i on- esponsi e ECF sigma ac o P dS [
14
,
23
,
41
]. Simila ly,
membe s o he g oups ECF203 and ECF234 a e possibly egula ed by Te R ep esso s (in he
case o g oup ECF203) o wo-componen sys ems (in he case o g oup ECF234) conse ed
in hei gene ic neighbo hoods [
7
]. SigH om Po phy omonas gingi alis (ECF114) is induced
in he p esence o oxygen in a SigH-independen manne , sugges ing ha i is also ac i a ed
by ansc ip ional egula ion [28].
3.2. ECF Sigma Fac o s Regula ed by Con o ma ional Changes
This mechanism o ac ion has been p oposed o SigC om M. ube culosis (ECF36),
al hough u he expe imen al da a will be equi ed o be ully es ablished (Figu e 2B).
This sigma ac o is in ol ed in i ulence du ing in ec ion and in coppe acquisi ion
unde me al-limi ing condi ions [
42
,
43
]. Despi e being highly exp essed du ing mos o
he M. ube culosis li e cycle, SigC is no o en ound in complex wi h he RNAP co e
enzyme, sugges ing ha he p o ein migh be no mally ansla ed in an inac i e o uns able
con o ma ion [
44
]. The ac ha
in i o
analyses showed ha bo h domains o he p o ein
a e able o in e ac , occluding he DNA binding si es o he sigma ac o , sugges s ha he e
is a con o ma ional change equi ed o SigC o be ac i e [45].
In . J. Mol. Sci. 2022,23, 3601 7 o 13
3.3. ECF Sigma Fac o s Regula ed by P o eolysis
This mechanism o egula ion, mainly ound in Ac inobac e ia, uses di e en pep idases—
such as Clp p o eases, sub ilases, o ca boxipep idases— o keep he sys em inac i e by
p o eoly ic deg ada ion o he sigma ac o .
To da e, only An A (ECF282) om S ep omyces albus has been shown expe imen ally
o exhibi his kind o p o eolysis- egula ed mechanism. The p o ein An A egula es pa
o he gene clus e in ol ed in he syn hesis o he bioac i e compound an imycin. This
egula o holds a C- e minal AA mo i (a di-alanine a he C e minus) which ac s as a
di ec a ge o he p o ease ClpXP o deg ade he p o ein (Figu e 2C), p e en ing i s
accumula ion and ansc ip ion o he genes unde i s con ol [
29
,
46
]. E en hough his
sigma ac o is ansc ip ionally egula ed by he LuxR ep esso FscRI,
in i o
s udies ha e
shown ha is he ac ion o he ClpXP p o ease which plays a majo ole on he condi ional
p esence o An A du ing di e en s ages o he li e cycle o S. albus [29].
The use o p o eoly ic enzymes o con ol he ac i i y o ECF sigma ac o s in he
absence o an an i-sigma ac o has also been p oposed o he membe s o he g oup ECF54
due o he conse ed p esence o p o eins holding ca boxypep idase, sub ilase, and caspase
He F associa ed wi h Tp s (CHAT) domains in hei gene ic con ex [7,47].
3.4. ECF Sigma Fac o s Regula ed by Phospho yla ion
A signi ican numbe o ECF sigma ac o s ha e been p edic ed o be egula ed by
phospho yla ion, an ac i a ion mechanism e y di e en om ha o canonical ECFs. This
p edic ion was based on mic osyn eny s udies, which e ealed ha hey a e encoded in he
p oximi y o a gene o an STPK [1,5,7].
One o he ECF sigma ac o s egula ed by phospho yla ion is Ec K om Xan homonas
ci i [
48
]. This sigma ac o (included in g oup ECF43) is encoded nex o he STPK PknS,
in a egion ha encodes a ype VI sec e ion sys em (T6SS) equi ed o p o ec cells om
p eda ion by he amoeba Dic yos elium discoideum. In his s udy, i has been demons a ed
ha PknS is equi ed o induce he exp ession o he T6SS, and ha a phosphomime ic
mu a ion in Th 51 ( he esidue p edic ed o be phospho yla ed) by a glu amic acid is
able o up egula e he exp ession o he T6SS in a
∆
pknS mu an . Acco ding o hese
da a, i has been pos ula ed ha PknS, in he p esence o D. discoideum, ac i a es Ec K by
phospho yla ion, which up egula es he T6SS o esis p eda ion [48].
Mo e ecen ly, i has been demons a ed ha Ec P om Vib io pa ahaemoly icus (ECF43)
is ac i a ed by phospho yla ion by he STPK PknT in esponse o polymyxin o egula e he
exp ession o a egulon ha con e s esis ance o his an ibio ic [
30
]. Con a y o canonical
ECF sigma ac o s, Ec P is in insically inac i e (Figu e 2D). This inac i i y elies on he
ac ha i lacks a DAED mo i in he
σ
2.2 egion ( his name e e s o he amino acids
ound in his mo i ), which con ains he nega i ely cha ged esidues usually equi ed
o in e ac wi h posi i ely cha ged esidues o he
β
’ subuni o he RNAP [
49
–
52
]. In
con as , Ec P shows in his egion an STTA mo i (also e e ed o he esidues ound in
he same posi on), in which he second Th is he esidue phospho yla ed by PknT [
30
].
This phospho yla ion p o ides he nega i e cha ge equi ed o in e ac ion wi h he co e
RNAP. In his signal- ansduc ion pa hway, i is hypo hesized ha PknT somehow senses
he s ess o igina ed by polymyxin, inducing he kinase ac i i y and he phospho yla ion
o Ec P o exp ess genes in ol ed in polymyxin esis ance.
As men ioned abo e, V. pa ahaemoly icus Ec P is included in g oup ECF43 o sigma
ac o s, and all membe s o his g oup lack he DAED mo i in he
σ
2.2 egion, indica ing
ha all o hem unc ion in a simila manne , being ac i a ed by phospho yla ion by an
STPK encoded in he p oximi y o he sigma ac o gene [
7
]. In ac , Iye e al. (2020) also
demons a ed ha ano he ECF43 sigma ac o om Hyphomonas nep unium is ac i a ed
in a simila manne by an STPK [
30
]. Acco ding o he mos ecen classi ica ion o ECF
sigma ac o s, a o al o six g oups (including ECF43) ha e been p oposed o be ac i a ed
by phospho yla ion, because hey a e no usually co-exp essed wi h an an i-sigma ac o ,
bu wi h an STPK [
7
]. Howe e , ECFs om g oups di e en om ECF43 may unc ion in a
In . J. Mol. Sci. 2022,23, 3601 8 o 13
di e en manne as hey exhibi a DAED mo i (o a e y simila sequence wi h nega i ely
cha ged esidues) o in e ac wi h he RNAP. Ne e heless, bioin o ma ic analyses ha e
shown ha ei he Se o Th esidues appea in some g oups in o a ound his mo i ha
could be he a ge o an STPK [
30
]. Cha ac e iza ion o mo e ECF sigma ac o s o di e en
g oups pos ula ed o be egula ed by phospho yla ion will be necessa y o elucida e hei
mechanisms o ac ion, which may di e om ha epo ed o Ec P.
3.5. ECF Sigma Fac o s wi h Regula o y Ex ensions
These ECF sigma ac o s, while lacking a egula o y an i-sigma ac o , conse e a N-
e minal o a C- e minal ex ension ha ypically akes o e he senso y and/o egula o y
ole o hose missing p o eins. In some cases, hese egula o y ex ensions seem indeed o
ha e been o igina ed by an ancien ansla ional usion wi h hei an i-sigma ac o . ECF
sigma ac o s wi h egula o y C- e minal ex ensions we e i s desc ibed in Myxococcus
xan hus [
32
] as well as in Bacillus licheni o mis and R. sphae oides [
53
]. A e ha , many o he
ECF sigma ac o s wi h egula o y ex ensions ha e been iden i ied bioin o ma ically [
6
,
7
,
25
]
o expe imen ally demons a ed [
31
,
33
,
54
]. The la es classi ica ion o ECF sigma ac o s
shows ha membe s o a leas 26 ECF phylogene ic g oups p esen N- o C- e minal
ex ensions p edic ed o ha e a key egula o y ole [
7
]. To da e, h ee main molecula
mechanisms go e ning his egula ion ha e been desc ibed: ac i a ed by con o ma ional
change, ac i a ed by p o ein in e ac ion, and ac i a ed by p o eolysis. These mechanisms
comp ehend 11 di e en ECF sigma ac o g oups, while he o he 15 emain o be s udied
and may show addi ional egula o y mechanisms (Table 2).
3.5.1. Ac i a ed by Con o ma ional Change
The C- e minal ex ension o hese p o eins is a s uc u al domain p edic ed o in e ac
wi h he
σ
2 and
σ
4 domains o he ECF sigma ac o . This ex ension, when binding o
speci ic sub ac s (likely small molecules), modula es he con o ma ion o he ECF sigma
ac o , allowing p omo e ecogni ion, and pe mi ing he ECF o ca y ou i s ac i i y. The
ECFs ollowing his egula ion ha e been ecen ly assigned o g oups ECF41 and ECF238.
Co E and Co E2 om M. xan hus (ECF238) ep esen he bes unde s ood g oup o
ECF sigma ac o s wi h C- e minal ex ensions. These p o eins hold a cys eine- ich domain
(CRD) in he C- e minus and a CXC mo i in he linke be ween
σ
2 and
σ
4, bo h essen ial
o ac i i y (Figu e 3A) [
2
,
32
,
33
]. The e-classi ica ion o Co E-like sigma ac o s in o he
new ECF238 g oup aises some challenges compa ed o hei p e ious classi ica ion in o he
o me ECF44 g oup. Fi s o all, Co E— he ounding membe o hese egula o s—is now
unclassi ied unde he new classi ica ion c i e ia. Second, se e al o he signa u e ea u es
o he Co E-like sigma ac o s, like he p esence o he CXC mo i , a e only conse ed o
he membe s o he o me ECF44 g oup, bu no o he emaining ~85% membe s o he
new ECF238 g oup. The di icul ies o accu a ely assigning Co E-like sigma ac o s in o his
g oup (like in he case o Co E), oge he wi h he lack o uni o mi y wi hin he new ECF238
g oup, a gues o a ca e ul e-examina ion o his g oup and p obably o a es i u ion o
he o me ECF44 phylogene ic g oup.
Despi e hei simila i ies, bo h cha ac e ized Co E-like p o eins exhibi clea di e -
ences. Co E, which is in ol ed in coppe homeos asis, unde goes an ac i a ion/inac i a ion
p ocess dependen on he coppe edox s a e, in which Co E is ac i a ed by Cu
2+
, while i is
quickly inac i a ed by Cu
+
due o he s ong educing condi ions o he cy oplasm [
32
,
55
].
Howe e , Co E2 esponds o Cd
2+
and Zn
2+
, and consequen ly, he inac i a ion is obse ed
in he absence o hese me als. The Cys dis ibu ion o hei C- e minal ex ensions has been
demons a ed o be esponsible o hei me al speci ici y and he ype o esponse [
32
,
33
].
Due o he abundance o cys eine esidues in hei C- e minal ex ensions, o he ECF sigma
ac o s—such as he membe s o g oups ECF287 and ECF288—ha e been sugges ed o
ha e a simila egula o y mechanism o he Co E-like sigma ac o s.
In . J. Mol. Sci. 2022,23, 3601 9 o 13
Figu e 3.
An i-sigma ac o s go e ned by egula o y ex ensions. ECF sigma ac o s a e depic ed in
blue, whe eas hei egula o y ex ensions and o he egula o y p o eins a e depic ed in g een and
pink, espec i ely. (
A
) The Co E C- e minal ex ension (CRD) om M. xan hus esponds o coppe
edox s a e; (
B
) Regula ion by p o ein in e ac ion o ECF42; (
C
) Iu Y om P. pu ida is deg aded
by he ca boxypep idase P c and he me allop o ease RseP. OM: ou e memb ane; CM: cy oplas-
mic memb ane.
On he o he hand, ECF sigma ac o s om he ECF41 g oup, exempli ied by SigJ and
SigI om M. ube culosis, as well as Ec 41Bli om B. licheni o mis and R. sphae oides, and
RpoE10 om Azospi illum b asilense, hold a C- e minal ex ension wi h a SnoaL-like domain.
Con a y o he Co E-like ECF sigma ac o s, he C- e minal ex ensions o hese p o eins
play an inhibi o y ole by con ac ing a mo i in he linke egion, p e en ing i s binding
o he RNAP co e enzyme [
31
,
53
,
56
]. Pa o he C- e minal ex ension, howe e , seems
o be equi ed o he p ope con o ma ion o he ac i e ECF sigma ac o , sugges ing an
ac i a ion mechanism media ed by a con o ma ional change a he han by p o eoly ic
clea age o he C- e minal ex ension [
25
,
53
,
54
,
57
]. O he ECF sigma ac o g oups—such as
ECF56, ECF205, ECF294, and ECF295—con ain C- e minal ex ensions o 120–150 esidues
bea ing SnoaL-like domains, al hough hei egula o y unc ions emain o be elucida ed [
7
].
3.5.2. Ac i a ed by P o ein In e ac ion
Like ECF sigma ac o s desc ibed in he p e ious sec ion, hese p o eins a e expec ed
o unde go a con o ma ional change media ed by hei C- e minal ex ensions. Howe e ,
unlike membe s o ECF238 and ECF41, his con o ma ional change is no p edic ed o be
p omp ed by he di ec binding o he ligand o hei C- e minal ex ensions, bu by he
in e ac ion wi h o he p o eins.
This in e ac ion-dependen egula ion has been p oposed o membe s o he g oup
ECF42. Among hem, he bes s udied a e S en_0747, S en_7131, and S en_4377, om
S ep omyces enezuelae [
58
], and ECF-10 om Pseudomonas pu ida, which con e s sensi i -
i y o oxida i e s ess and an ibio ics, and is homologous o ECF sigma ac o s o nea
4000 o he Pseudomonas s ains [
23
,
59
]. These p o eins hold a C- e minal ex ension o ap-
p oxima ely 200 esidues ich in e a icopep ide epea (TPR) domains, usually in ol ed
in p o ein–p o ein in e ac ions, which is comple ely essen ial o he ac i i y o hese p o-
eins [
6
,
31
,
58
]. As men ioned abo e, i is p edic ed ha he in e ac ion o he C- e minal
ex ension wi h ano he p o ein esul s in a con o ma ional change in he ECF sigma ac-
o ha ac i a es/inac i a es i (Figu e 3B). Only he p oximal egion o he C- e minal
ex ension is p edic ed o in e ac wi h he
σ
4 domain o he sigma ac o [
31
], whe eas
he in e ac ion pa ne (s) o he emaining ex en o he p o ein emains o be iden i ied.
Ne e heless, he conse ed p esence o a YCII- ela ed domain con aining p o ein in hei
gene ic neighbo hood, and he ac ha hese domains can also be ound used o o he ECF
sigma ac o s (like in he case Q9A8M4_CAUVC om Caulobac e ib ioides), sugges ing
ha his conse ed p o ein is also in ol ed in hei egula o y mechanism.
A simila mechanism o egula ion has been sugges ed o some membe s o he
g oup ECF57 o sigma ac o s, which exhibi s a p e alence o WD40-like epea s, ypically
in ol ed in p o ein–p o ein in e ac ions, in hei C- e minal ex ensions [
6
]. The p esence o
wo conse ed cys eine esidues ha po en ially link he
σ
2 and he
σ
4 domains sugges
ha he e is a s ong con o ma ional change in ol ed in he ac i a ion o hese p o eins.
