Role o Cen al Me abolism in he Osmoadap a ion o he
Halophilic Bac e ium Ch omohalobac e salexigens
*
□
S
Recei ed o publica ion, Ma ch 18, 2013, and in e ised o m, Ap il 8, 2013 Published, JBC Pape s in P ess, Ap il 24, 2013, DOI 10.1074/jbc.M113.470567
José M. Pas o
‡1,2
, Vicen e Be nal
‡1,3
, Manuel Sal ado
§2
, Mon se a A gandoña
§4
, Ca men Va gas
§
,
Laszlo Csonka
¶5
, Ángel Se illa
‡6
, José L. Ibo a
‡
, Joaquín J. Nie o
§
, and Manuel Cáno as
‡7
F om he
‡
Depa amen o de Bioquímica y Biología Molecula B e Inmunología. Facul ad de Química, Campus Regional de
Excelencia In e nacional “Campus Ma e Nos um,” Uni e sidad de Mu cia, 30100 Mu cia, Spain, he
§
Depa amen o de
Mic obiología y Pa asi ología, Uni e sidad de Se illa, 41012 Se ille, Spain, and he
¶
Depa men o Biological Sciences, Pu due
Uni e si y, Wes La aye e, Indiana 47907-2064
Backg ound: Ch omohalobac e salexigens syn hesizes and accumula es ec oines.
Resul s: High a io o he anaple o ic and ca abolic luxes in ol ed in ec oines syn hesis suppo s high biosyn he ic luxes a high
salini y and leads o me aboli e o e low a low salini y.
Conclusion: E olu ion op imized he me abolism o C. salexigens o suppo high p oduc ion o ec oines.
Signi icance: Me abolic adap a ions in a compa ible solu e-accumula ing halophile a e desc ibed o he i s ime.
Bac e ial osmoadap a ion in ol es he cy oplasmic accumu-
la ion o compa ible solu es o coun e ac ex acellula osmo-
la i y. The halophilic and highly halo ole an bac e ium Ch o-
mohalobac e salexigens is able o g ow up o 3 MNaCl in a
minimal medium due o he de no o syn hesis o ec oines. This is
an osmo egula ed pa hway ha bu dens cen al me abolic
ou es by quan i a i ely d awing o TCA cycle in e media ies.
Consequen ly, me abolism in C. salexigens has adap ed o sup-
po his biosyn he ic ou e. Me abolism o C. salexigens is mo e
e icien a high salini y han a low salini y, as e lec ed by lowe
glucose consump ion, lowe me aboli e o e low, and highe
biomass yield. A low salini y, by-p oduc s (mainly glucona e,
py u a e, and ace a e) accumula e ex acellula ly. Using
[1-
13
C]-, [2-
13
C]-, [6-
13
C]-, and [U-
13
C
6
]glucose as ca bon
sou ces, we we e able o de e mine he main cen al me abolic
pa hways in ol ed in ec oines biosyn hesis om glucose. C.
salexigens uses he En ne -Doudo o pa hway a he han he
s anda d glycoly ic pa hway o glucose ca abolism, and anaple-
o ic ac i i y is high o eplenish he TCA cycle wi h he in e -
media ies wi hd awn o ec oines biosyn hesis. Me abolic lux
a ios a low and high salini y we e simila , e ealing a ce ain
me abolic igidi y, p obably due o i s specializa ion o suppo
high biosyn he ic luxes and pa ially explaining why me abolic
yields a e so highly a ec ed by salini y. This wo k ep esen s an
impo an con ibu ion o he elucida ion o speci ic me abolic
adap a ions in compa ible solu e-accumula ing halophilic
bac e ia.
Halophilic mic oo ganisms demand ela i ely high sal con-
cen a ions o g ow. Because o he di e si y o en i onmen s
whe e hey can h i e, hei physiology is widely a ied. The
me abolic di e si y o halophilic and halo ole an mic oo gan-
isms is condi ioned by he adap a ion o speci ic en i onmen al
niches (especially e iden in he case o alkalophilic, me ha-
no ophic, o he mophilic halophilic bac e ia) and also by
osmoadap a ion mechanisms ha hese mic oo ganisms de el-
oped o cope wi h salini y (1–3). As a consequence, he p e-
e ed me abolic pa hways used o assimila e ca bon sou ces
a e also di e se.
S a egies o osmoadap a ion can be oughly classi ied in wo
main ypes. The “sal -in” s a egy, which consis s o he accu-
mula ion o K
⫹
and Cl
⫺
in he cy oplasm o he cells, is used by
ex emely halophilic ae obic a chaea, halophilic e men a i e
bac e ia, and he ex emely halophilic bac e ium Salinibac e
ube (4–6). The “o ganic solu es-in” s a egy, which in ol es
he accumula ion o o ganic “compa ible” solu es, is used by a
la ge a ie y o o ganisms, including all mesophilic bac e ia,
halophilic algae, halophilic me hanogenic a chaea, and halo ol-
e an and halophilic ae obic bac e ia (7). Among he halophilic
eubac e ia ha use he o ganic solu es-in s a egy, s ic ae o-
biosis is mo e equen because compa ible solu e syn hesis is
ene ge ically and me abolically a e y demanding p ocess (4).
Compa ible solu es belong o a ew chemical amilies: suga s
(suc ose and ehalose), polyols (glyce ol, glucosylglyce ol,
mannosylglyce ol, and a abi ol, among o he s), amino acids
(glu amine and de i a i es, p oline, alanine), qua e na y
amines (be aines and choline), and ec oines (ec oine and
␤
-hy-
d oxyec oine). Ec oine is one o he mos widely dis ibu ed
*This wo k was suppo ed in pa by Fondo Eu opeo de Desa ollo Regional
unds, Minis e io de Ciencia e Inno ación (Spain) P ojec s BIO2008-04502-
01, BIO2011-29233-C02-01, and BIO2011-22833, Jun a de Andalucía
(Spain) G an P08-CVI-03724, and Spanish Na ional Ne wo k on Ex emo-
philic Mic oo ganisms G an BIO2011-12879-E.
□
S
This a icle con ains supplemen al Ma e ials and Me hods, Tables S1–S4,
Figs. S1–S8, and addi ional e e ences.
1
Bo h au ho s con ibu ed equally o his wo k.
2
Recipien o Fo mación de P o eso ado Uni e si a io and Fo mación de Pe -
sonal In es igado ellowships om MICINN (Spain).
3
Recipien o a pos -doc o al con ac om Uni e sidad de Mu cia (P og ama
P opio). To whom co espondence may be add essed: G upo de Bioen-
e gía, Di ección de Tecnología, Cen o de Tecnología de Repsol, Ca e e a
A-5, Km 18, 28935 Mós oles-Mad id, Spain. Tel.: 34-868-887393; Fax:
34-868-884148; E-mail: [email p o ec ed].
4
Recipien o a pos -doc o al con ac om Jun a de Andalucía.
5
Suppo ed by Na ional Science Founda ion Awa d IOS-1054977.
6
Recipien o a pos -doc o al con ac om he P og ama Juan de la Cie a
(Spain).
7
To whom co espondence may be add essed. Tel.: 34-868-887393; Fax:
34-868-884148; E-mail: [email p o ec ed].
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 24, pp. 17769–17781, June 14, 2013
© 2013 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc. Published in he U.S.A.
JUNE 14, 2013•VOLUME 288•NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 17769
a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om
compa ible solu es. Hyd oxyec oine is o en syn hesized a
lowe amoun s oge he wi h ec oine by many ec oine-p oduc-
ing species (8). When p esen in he medium, ei he hese com-
pounds o hei p ecu so s can be aken up om he en i on-
men . When cells a e g owing in media lacking compa ible
solu es, hese compounds can only be accumula ed by de no o
syn hesis (9).
Ch omohalobac e salexigens DSM 3043 ( o me ly Halomo-
nas elonga a DSM 3043) is a halophilic
␥
-p o eobac e ium o
he amily Halomonadaceae (10). I has one o he wides salin-
i y anges o g ow h ound in na u e (10, 11). In C. salexigens,
osmoadap a ion is mainly achie ed by he accumula ion o
ec oine and hyd oxyec oine (designa ed oge he as “ec oines”)
(9). The biosyn he ic pa hway o ec oines was es ablished in H.
elonga a and C. salexigens (11–15).
The a ailabili y o he genomic sequence o C. salexigens (16)
p esen s an oppo uni y o he unde s anding o he cha ac-
e is ic me abolic ea u es o his halophile and hei ele ance
o osmoadap a ion. Du ing ac i e g ow h, me aboli e luxes
a e subs an ially di ec ed owa d biosyn he ic pa hways, d ain-
ing in e media ies o cen al me abolism. The e o e, he pe -
ec coupling and e iciency o he me abolic pa hways linking
ca bon sou ces o he end p oduc s (in ou case, compa ible
solu es) is c ucial. The e a e wo majo pa hways o he ca ab-
olism o suga s o py u a e: glycolysis (Embden-Meye ho
pa hway) (EM)
8
and he En ne -Doudo o (ED) pa hway (17).
The physiological ele ance o hese pa hways o he g ow h o
C. salexigens is no known (16, 18, 19). The syn hesis o ec oines
consumes ace yl-CoA, which is p oduced by oxida i e deca -
boxyla ion o py u a e, and oxaloace a e (OAA), which is an
in e media e in he TCA cycle and has o be eplenished by
anaple o ic pa hways (16). The lux a ios be ween anaple o ic
and ca abolic pa hways a e highly ele an o me abolic
adap a ion.
Me abolic s udies in halophilic and halo ole an bac e ia a e
sca ce, especially i a ocus on he biosyn hesis o compa ible
solu es is sough . In his wo k, we gained new insigh s in o he
ole o cen al me abolism in he osmoadap a ion o C. salexi-
gens. Using iso ope label acing, we analyzed he pa hways o
glucose ca abolism and how cen al me abolism copes wi h he
high me abolic bu den caused by ec oines biosyn hesis. Ou
esul s show how he adap a ions de eloped by his bac e ium
a ec me abolic e iciency a di e en salini ies and ep esen a
s ep u he in he unde s anding o he physiology o halophilic
and halo ole an bac e ia.
MATERIALS AND METHODS
Bac e ial S ains and Cul u es
C. salexigens CHR61, a i ampicin- esis an spon aneous
mu an o C. salexigens DSM 3043
T
, was used h oughou his
s udy. Fo ec oine p oduc ion and o cha ac e iza ion o me -
abolic pa hways, he s ain was g own in minimal medium M63
(pH 7.2) con aining 16.3 g/li e KH
2
PO
4
, 4.2 g/li e KOH, 2
g/li e (NH
4
)
2
SO
4
, 39.5 mg/li e MgSO
4
䡠7H
2
O, 0.5 mg/li e
FeSO
4
䡠7H
2
O. M63 was supplemen ed wi h 35.0, 43.8, 146.0, o
175.2 g/li e NaCl (co esponding o 0.6, 0.75, 2.5, o 3 M). As a
ca bon sou ce, 20 mMglucose was used. Ae obic 100-ml ba ch
cul u es we e g own in 0.5-li e lasks a 37 °C on a o a y
shake a 210 pm.
Cul u es we e s a ed om ozen 20% glyce ol s ocks. P e-
cul u es we e g own in SW-2 medium (con aining 2% (w/ ), o
0.3 M, o al sal s) composed o 15.6 g/li e NaCl, 4.07 g/li e
MgSO
4
䡠7H
2
O, 2.6 g/li e MgCl
2
䡠6H
2
O, 0.4 g/li e KCl, 67
mg/li e CaCl
2
䡠2H
2
O, 47 mg/li e NaB , and 13 mg/li e
NaHCO
3
(20). M63 cul u es we e inocula ed o an ini ial
abso bance (A
600
) o 0.025 wi h an exponen ial phase p ecul-
u e g own o e nigh in SW-2 medium. Glyce ol s ocks, solid
cul u e media, and p ecul u es we e supplemen ed wi h i am-
picin o a inal concen a ion o 25
␮
g/ml.
Analy ical P ocedu es
Cell G ow h—To measu e cell concen a ion, cells we e
esuspended in a NaCl solu ion (0.6 o 3.0 M), and abso bance
was measu ed a 600 nm (No aspec Plus Visible Spec opho-
ome e , Ame sham Biosciences). A
600
and g am o d y cell
weigh (g
DCW
) we e co ela ed o he s ain used, acco ding o
he ollowing empi ical equa ions: g
DCW
/li e ⫽0.597䡠A
600
( o
0.6 Mg own cul u es), g
DCW
/li e ⫽0.557䡠A
600
( o 0.75 M
g own cul u es), and g
DCW
/li e ⫽0.532䡠A
600
( o 2.5 and 3 M
g own cul u es).
De e mina ion o Ex acellula O ganic Acids—Ex acellula
o ganic acids we e de e mined by ion exchange ch oma og a-
phy. Ace a e was analyzed in a Shimadzu LC-10 HPLC ins u-
men (Shimadzu Scien i ic Ins umen s, Columbia, MD),
equipped wi h di e en ial e ac i e index and diode a ay
(UV) de ec o s (Shimadzu Scien i ic Ins umen s, Columbia,
MD). A ca ion exchange HPX-87H column (Bio-Rad) was used
o he sepa a ion o o ganic acids. The mobile phase was 5 mM
H
2
SO
4
a a 0.5 ml䡠min
⫺1
low a e and 45 °C. Glucona e (m/z
195), py u a e (m/z87), lac a e (m/z89), and ci a e (m/z191)
we e measu ed using HPLC-MS. Analysis was pe o med wi h
an Agilen 1200 se ies HPLC ins umen (Agilen Technolo-
gies, San a Cla a, CA) coupled o an Agilen 6120 single qua-
d upole mass spec ome e wi h o hogonal elec osp ay ioni-
za ion sou ce. The mass spec ome e was ope a ed in he
nega i e elec osp ay ioniza ion mode, using he SCAN mode
a a ange o m/z50–300, whe eas he selec ed ion moni o ing
mode was used o he m/zo each compound. The ion sp ay
ol age was se a 3500 V. Ni ogen wi h a lux o 12 li e s/min
was used as he shea h gas (40 p.s.i.) and he auxilia y gas. The
ion ans e capilla y was hea ed o 350 °C. The agmen a ion
ol age was se a 70 V. Sepa a ion was ca ied ou on an injec-
ion olume o 10
␮
l using he same column and condi ions as
he p e ious me hod, subs i u ing 5 mMH
2
SO
4
wi h 0.1% o -
mic acid. Da a we e acqui ed by a PC using he Agilen Chem-
s a ion so wa e.
Iso opic Labeling S udies and NMR Spec oscopy—Fo he
labeling expe imen s, cells we e g own in 100 ml o M63
medium in he p esence o iso opically labeled glucose (Co ec-
8
The abb e ia ions used a e: EM, Embden-Meye ho ; ED, En ne -Doudo-
o ; OAA, oxaloace a e; Gdh, glu ama e dehyd ogenase; Pc, py u a e
ca boxylase; Ppc, phosphoenolpy u a e ca boxylase; Icdh, isoci a e
dehyd ogenase; Cs, ci a e syn hase; P k, 6-phospho uc okinase; Gdh,
glu ama e dehyd ogenase; PEP, phosphoenolpy u a e; Pdh, py u a e
dehyd ogenase.
Cen al Me abolism and Osmoadap a ion in C. salexigens
17770 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 24•JUNE 14, 2013
a FAC BIOLOGIA/BIBLIOTECA on June 20, 2016h p://www.jbc.o g/Downloaded om
Ne , Voisins-Le B e onneux, F ance). Iso opically labeled sub-
s a es used we e 100% [1-
13
C]-, [2-
13
C]-, [6-
13
C]glucose, o
20% [U-
13
C
6
]glucose. Cul u es we e ha es ed in he mid o la e
exponen ial phase (A
600
1.5 o 3), and cells we e sepa a ed om
supe na an s by cen i uga ion (16,000 ⫻g, 15 min, 4 °C).
Supe na an s we e concen a ed by lyophiliza ion, edis-
sol ed in 1 ml o deu e a ed me hanol (Sigma) and used o he
iden i ica ion o ex acellula by-p oduc s.
13
C nuclea magne ic
esonance (
13
C NMR) spec a we e eco ded on a B ucke AV200
spec ome e a 200 MHz and 25 °C, wi h a elaxa ion ime o 1.5 s.
Signals o py u a e, glucona e, and ace a e we e assigned by com-
pa ison wi h p e iously published chemical shi s (Spec al Da a-
base o O ganic Compounds) and con i med by compa ison wi h
13
C NMR spec a o pu e compounds.
Compa ible solu es (ec oines, glu ama e, and ehalose) and
memb ane lipids we e ex ac ed om he cell pelle s by a a i-
a ion o he p o ocol desc ibed by Ga cía-Es epa e al. (12). The
aqueous phase was used o he analysis o compa ible solu es,
and he chlo o o m phase o memb ane lipids. Spec a we e
eco ded a 25 °C using B ucke AV400 and B ucke AV600
spec ome e s a 400 and 600 MHz, espec i ely, and a elax-
a ion ime o 3 s. Peak a eas we e in eg a ed o ela i e
quan i ica ion.
Spec opho ome ic De e mina ion o Glucose and Ammonia
Consump ion—Glucose was assayed by a glucose (hexokinase)
assay ki (GAHK20, Sigma). Ammonium was assayed by an enzy-
ma ic assay ki (11112732035, om R-Biopha m, Da ms ad , Ge -
many). Ki s we e used acco ding o he ecommenda ions o he
manu ac u e s. Measu emen s we e pe o med in a 96-well
mic opla e eade Syne gy HT (Bio-Tek, Winooski, VT).
Enzyme Assays
Enzyme assays we e op imized o he condi ions, media, and
he mic oo ganisms used in his wo k. Measu emen s we e ca -
ied ou in a 96-well mic opla e eade Syne gy HT (Bio-Tek,
Winooski, VT). A uni o enzyme ac i i y was de ined as mic o-
moles o subs a e consumed o p oduc o med pe min and
was no malized o millig ams o p o ein (uni s䡠mg
⫺1
).
In each case, eac o bulk samples we e wi hd awn, and cells
we e cen i uged (16,000 ⫻g, 15 min, 4 °C) and esuspended in
65 mMphospha e bu e (pH 7.5). Cells we e sonica ed on ice
wi h a 3-mm diame e p obe using a Vib a Cell VC 375 ul a-
sonic p ocesso (Sonics Ma e ials, Danbu y, CT) and cen i-
uged (16,000 ⫻g, 20 min, 4 °C). The supe na an (cell- ee
ex ac ) was used o subsequen ac i i y measu emen s. P o-
ein concen a ion in cell- ee ex ac s was de e mined by he
bicinchoninic acid (BCA) me hod (BCA P o ein Assay ki ,
Pie ce).
The p o ocols o he assessmen o he ac i i y o 6-phos-
pho uc okinase (P k) (21), glucose-6-phospha e dehyd ogen-
ase (21), ci a e syn hase (Cs) (21), NADP
⫹
/NAD
⫹
-dependen
isoci a e dehyd ogenase (Icdh) (22), py u a e ca boxylase (Pc)
(23), phosphoenolpy u a e ca boxylase (Ppc) (23), isoci a e
lyase (22), malic enzyme (24), aspa a e amino ans e ase (25),
NADPH and NADH-dependen glu ama e dehyd ogenase
(Gdh) (26) we e op imized o C. salexigens (see supplemen al
ma e ial).
In Silico Analysis o P o ein Homology
The comple ely sequenced and anno a ed genome o C.
salexigens is a ailable on line. Howe e , he anno a ions ha e
been made by au oma ed homology s udies o ORFs om many
mic oo ganisms, and he e may be some inco ec assignmen s
o gaps in he in o ma ion. To analyze in de ail he ORF assign-
men s o he me abolic pa hways mo e ele an o his wo k,
we compa ed he me abolic econs uc ion made by A es e al.
(18) wi h in o ma ion a ailable a genome sequence-based
da abases, such as he Kyo o Encyclopedia o Genes and
Genomes (KEGG) (27), and Me aCyc (28), which a e suppo ed
by expe imen al da a. Gene and p o ein sequences we e com-
pa ed using BLAST (29). Analyses o domains (Conse ed
Domain Da abase, www.ncbi.nlm.nih.go ), p o ein localiza ion
and opology (Signal P 4.0 se e ), and genomic con ex we e
also pe o med.
P edic ion o he Fa es o Iso opic Labels
[1-
13
C]-, [2-
13
C]-, and [6-
13
C]glucose we e selec ed speci i-
cally o in e oga ing he ela i e impo ance o di e en pa h-
ways o cen al me abolism, as desc ibed p e iously (30, 31).
The pa e ns o inco po a ion o he iso opic label om glucose
in o py u a e and ace yl-CoA ia glycolysis o he En ne -Dou-
do o pa hway and hen in o ec oines we e p edic ed.
RESULTS
High Salini y Fa o s Biomass and Ec oine P oduc ion by C.
salexigens
To s udy he e ec o salini y on he me abolism o C. salexi-
gens, he p oduc ion o biomass and ec oines was de e mined in
cul u es g own wi h glucose as he sole ca bon sou ce a 0.6,
0.75, 2.5, and 3 MNaCl. Maximum biomass p oduc ion
inc eased wi h salini y up o 2.5 MNaCl (Table 1), whe eas
TABLE 1
G ow h and p oduc ion o ec oines o C. salexigens a di e en NaCl concen a ions
Cul u es we e g own a 37 °C in M63 minimal medium wi h 20 mMglucose and 30 mMammonium as he sole ca bon and ni ogen sou ces (see “Ma e ials and Me hods”
o de ails).
NaCl concen a ion
in medium
Maximum
biomass Y
Ec /X
a
Ec oines
p oduc ion a e
Hyd oxyec oine/ec oine
a io
g
DCW
䡠li e
⫺1
mmol䡠g
DCW
⫺1
mmol䡠(g
DCW
䡠h)
⫺1
0.6 M1.40 ⫾0.07 0.18 ⫾0.01 0.049 ⫾0.003 0.048 ⫾0.097
0.75 M1.76 ⫾0.03 0.45 ⫾0.01 0.127 ⫾0.004 0.106 ⫾0.030
2.5 M2.48 ⫾0.02 1.10 ⫾0.05 0.174 ⫾0.008 0.507 ⫾0.037
3M2.03 ⫾0.26 1.68 ⫾0.09 0.159 ⫾0.009 0.423 ⫾0.036
a
S oichiome ic coe icien o ec oines p oduc ion. Y
Ec /X
and ec oines p oduc ion a es we e de e mined in he exponen ial phase o cul u es. Maximum biomass and hy-
d oxyec oine o ec oine a io we e ob ained in he ea ly s a iona y phase.
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g ow h a e was op imal in minimal medium M63 wi h 0.75 M
NaCl (Fig. 1) (9, 12).
We epo ed p e iously ha he in acellula con en o ec o-
ines inc eases wi h salini y (12), in ag eemen wi h he ole o
ec oines in osmop o ec ion. The s oichiome ic coe icien s o
ec oines syn hesis om biomass (Y
Ec /X
) showed ha o al ec o-
ines con en was di ec ly p opo ional o salini y (Table 1 and
Fig. 2A). Howe e , ec oines p oduc ion a e was maximal a 2.5
MNaCl (Table 1). The hyd oxyec oine o ec oine a io
inc eased wi h salini y up o 2.5 MNaCl (Table 1 and Fig. 2A),
and an in e se co ela ion was obse ed be ween he ela i e
con en o ec oines and p o eins wi h inc easing sal concen-
a ion (Fig. 2B).
Consump ion o Ca bon and Ni ogen Sou ces
As he biosyn hesis o ec oines occu s a he expense o cen-
al me abolic in e media es, i is expec ed o signi ican ly bu -
den me abolism. To assess salini y-dependen me abolic
changes, nu ien s and by-p oduc s we e moni o ed a di e -
en salini ies.
Glucose is he a o i e ca bon sou ce o C. salexigens (32).
The s oichiome ic coe icien o glucose consump ion was
highly a ec ed by salini y, being highes a 0.6 MNaCl and
dec easing by 75% a 3 MNaCl (Fig. 2C). Ammonium consump-
ion was qui e di e en , because he s oichiome ic coe icien
o ammonium up ake emained p ac ically unal e ed ega d-
less o salini y (Fig. 2C). This is a ema kable inding, because, in
p inciple, he high p oduc ion o ec oines a high salini y
should lead o a highe demand o he ni ogen sou ce. To be -
e unde s and he o e all al e a ions o me abolism a di e en
salini ies, we calcula ed he ammonium o glucose consump-
ion mola a io. This pa ame e inc eased wi h salini y up o
2.5 MNaCl (Fig. 2C), and a posi i e co ela ion be ween he
speci ic ec oines p oduc ion a e, and he ammonium o glu-
cose consump ion a ios was obse ed (Fig. 2, Aand B).
O e all, ou indings show ha he slow down in g ow h and
me abolism a high sal concen a ions a o biomass p oduc-
ion, which is in ag eemen wi h he p e iously obse ed e ec
o sal concen a ions abo e 1.5 MNaCl (32). This unde sco es
ha he highe e iciencies o ca bon and ni ogen me abolism
a high salini y a e he consequences o he specializa ion o
cope wi h a highly demanding en i onmen .
Quan i ica ion o O ganic Acids Exc e ed by C. salexigens,
O e low Me abolism
The p esence o glucona e, ace a e, py u a e, and mino
amoun s o lac a e in supe na an s o cul u es g own a 0.6 and
0.75 MNaCl sugges ed a possible o e low me abolism. The
consump ion o glucose and ammonium and p oduc ion and
eu iliza ion o o ganic acids we e de e mined a h ee di e en
salini ies. A any sal concen a ion, glucose was he g ow h-
limi ing nu ien , because i s deple ion ma ked he en ance
in o s a iona y phase. In con as , a ound 15 mMammonium
was s ill p esen in he medium a he end o g ow h o each
cul u e (da a no shown). A 0.6 and 0.75 MNaCl, ace a e was
p oduced du ing he exponen ial phase o g ow h (Fig. 1, Aand
B), al hough a high salini y (2.5 MNaCl) ex acellula ace a e
was almos unde ec able (Fig. 1C). The speci ic a e o p oduc-
ion o py u a e p oduc ion du ing exponen ial g ow h
dec eased wi h sal concen a ions (Table 2). Ace a e and py u-
a e we e e-assimila ed once glucose was o ally deple ed, in
con as wi h glucona e, which accumula ed in he cul u e
medium du ing ea ly exponen ial phase o g ow h and was con-
sumed along wi h glucose in he mid- o-la e exponen ial phase
(Fig. 1). As desc ibed in Table 2, glucona e, py u a e, and ace-
a e we e he majo exc e ed p oduc s. P oduc ion a es o he
wo la e condi ions in e sely co ela ed o he sal concen a-
ion and he biomass yield. These indings sugges an inc eased
me abolic e iciency a high salini y.
The p esence o hese compounds in cul u es g own sug-
ges ed a possible o e low me abolism a low salini y. This
FIGURE 1. Fo ma ion o biomass (black ci cles) and ex acellula concen-
a ions o glucose (black squa es), glucona e (whi e ci cles), py u a e
(whi e squa es), and ace a e (whi e iangles) o ba ch cul u es g own in
20 mMglucose minimal medium wi h 0.6 MNaCl (A), 0.75 MNaCl (B), and
2.5 MNaCl (C).
Cen al Me abolism and Osmoadap a ion in C. salexigens
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could a ise om a limi ed ca abolism o glucose, leading o a
highe exc e ion o by-p oduc s due o he educed demand o
ec oines.
In Silico and in Vi o Analyses o Me abolic Pa hways Rela ed
o he Syn hesis o P ecu so s o Ec oines
In Silico Analysis
Accumula ion o compa ible solu es a high salini y
imposes a biosyn he ic bu den on cells. The abo e esul s
e lec ha osmoadap a ion in C. salexigens has implica ions
on me abolic pe o mance. C. salexigens genome has been
au oma ically anno a ed, and p elimina y analyses (16, 19)
and a i s genome-based me abolic econs uc ion (18) ha e
been published. To u he unde s and he in e play
be ween osmoadap a ion and me abolism, we c i ically
assessed a numbe o ou es in his me abolic ne wo k
ela ed o cen al me abolism, wi h emphasis on he pa h-
ways leading o p ecu so s o ec oines, as well as o me abo-
li es ound in supe na an s. Fo his pu pose, we pe o med
he ollowing: (i) homology s udies using he in o ma ion o
ela ed mic oo ganisms such as Pseudomonads, En e obac-
e ia, and halo ole an bac e ia a ailable in me abolic da a-
bases such as Me aCyc and KEGG (27, 28); (ii) analysis o
conse ed domains, p o ein localiza ion, and genomic con-
ex ; (iii) g ow h expe imen s wi h D-glucono-1,5-lac one,
D-glucona e, and 2-ke o-D-glucona e as ca bon sou ces, and
(i ) e iew o li e a u e da a.
Genome analysis e ealed in e es ing me abolic ea u es.
The ED pa hway, which is a ou e o he ca abolism o glucose
o py u a e (33), could be ope a i e in C. salexigens (supple-
men al Fig. S1). Typically, mic oo ganisms using he ED pa h-
way lack glycoly ic enzyme(s), such as P k (34, 35). In ac ,
unambiguous anno a ion o he gene encoding P k in C. salexi-
gens has been di icul (19). The e a e i e ORFs anno a ed in
he JGI websi e (genome.o nl.go ) as po en ial phospho uc-
okinases, al hough hey ha e low homology wi h bona ide P ks
(supplemen al Fig. S2 and supplemen al Table S1). The e is also
a pu a i e py ophospha e-dependen phospho uc okinase
(Csal1534). The eac ion ca alyzed by his enzyme is eadily
e e sible (36). Howe e , he gene o his enzyme om P opi-
onibac e ium euden eichii can only complemen uc ose-
1,6-bisphospha ase ( bp) mu a ions bu no phospho uc oki-
nase (p kA/p kB) mu a ions in Esche ichia coli (37), indica ing
ha i unc ions in he gluconeogenic di ec ion bu no in he
glycoly ic di ec ion. Signi ican ly, he analysis o he C. salexi-
gens genome also ailed o iden i y a clea -cu ep esen a i e o
a uc ose bisphospha ase (19), aising he possibili y ha
Csal1534, which has been anno a ed as Ppi-P k, may be a uc-
ose bisphospha ase.
Ou in silico analysis sugges s ha oxida ion o D-glucose o
D-glucona e h ough D-glucono-1,5-lac one could occu bo h
in he pe iplasm and he cy oplasm. This ag ees wi h ou ind-
ing o he ea ly accumula ion o D-glucona e in he g ow h
medium. C. salexigens was able o g ow wi h D-glucono-1,5-
lac one, D-glucona e, and 2-ke o-D-glucona e as he sole ca bon
sou ce (supplemen al Fig. S4), and a a ian o he 2-ke o-glu-
cona e loop desc ibed in Pseudomonas (38) was p edic ed o be
unc ional in C. salexigens (Fig. 3 and supplemen al Fig. S1).
FIGURE 2. E ec o NaCl concen a ion on p oduc ion o ec oines and consump ion o ca bon and ni ogen sou ces in C. salexigens.A, cellula con en s
o ec oine and hyd oxyec oine. B, ela i e con en o p o eins and ec oines, exp essed as pe cen age o o al pool o ec oines plus p o eins. The sum o hese
pools was app oxima ely cons an h oughou all condi ions es ed (0.423 ⫾0.055 g/g
CDW
). C, s oichiome ic coe icien o glucose (da k ba s) and ammonium
consump ion (ligh ba s). Mola a io o ammonium o glucose u iliza ion is deno ed by ci cles. Cul u es we e g own a 37 °C in M63 minimal medium wi h 20
mMglucose and 30 mMammonium as he sole ca bon and ni ogen sou ces, espec i ely. See he ex o de ails.
TABLE 2
Speci ic consump ion/p oduc ion a es o he main ex acellula me aboli es
Cul u es we e g own in glucose/M63 minimal medium. See unde “Ma e ials and Me hods” o de ails. All a es we e calcula ed in he ea ly exponen ial phase o g ow h
and (excep o lac a e p oduc ion) a e exp essed in mmol䡠g
CDW
⫺1
䡠h
⫺1
.
[NaCl] Glucose Ammonium Glucona e Py u a e Lac a e
a
Ace a e
M
0.6 14.28 ⫾1.28 3.73 ⫾2.87 0.87 ⫾0.31 2.25 ⫾1.20 0.00 ⫾0.00 0.43 ⫾0.17
0.75 11.73 ⫾3.24 6.46 ⫾0.75 1.14 ⫾0.38 1.66 ⫾0.51 13.2 ⫾9.5 0.53 ⫾0.20
2.5 2.1 ⫾0.18 2.48 ⫾0.40 0.97 ⫾0.47 0.30 ⫾0.17 0.00 ⫾0.00 0.02 ⫾0.01
a
Lac a e p oduc ion a es a e in
␮
mol䡠g
CDW
⫺1
䡠h
⫺1
.
Cen al Me abolism and Osmoadap a ion in C. salexigens
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C. salexigens possesses genes speci ying pu a i e Pc, Ppc, and
OAA deca boxylase (supplemen al Table S1). These enzymes
in e con e py u a e, phosphoenolpy u a e, and OAA and
could ha e a ole in suppo ing high ec oine biosyn he ic luxes
by eplenishing OAA needed o he TCA cycle (Fig. 3).
The obse ed p oduc ion o ace a e is di icul o explain in
he ligh o he cu en genome anno a ion and knowledge o
me abolic pa hways. Ace a e me abolism in C. salexigens is
qui e di e en om ha o Pseudomonas and E. coli. The main
ou e o ace a e p oduc ion in P. ae uginosa, E. coli, and
ela ed species is he phospho ansace ylase ace a e kinase
pa hway (39), which is no p esen in C. salexigens. Ace a e
p oduc ion in he la e bac e ium could in ol e he o ma-
ion o he high ene gy in e media e ace yl phospha e
h ough an al e na i e ou e. Csal1010 is anno a ed as a sol-
uble py u a e oxidase. This FAD-dependen enzyme deca -
boxyla es py u a e, p oducing ace a e (28). The ca abolism
o ec oines also yields ace a e (40); in ac , con inuous syn-
hesis and deg ada ion o ec oines a low salini y could
explain he highe ace a e o e low.
Rega ding ni ogen me abolism, he e is one copy o genes
o alanine amino ans e ase, L-alanine dehyd ogenase, glu a-
ma e syn hase, and glu ama e dehyd ogenase in he C. salexi-
gens genome. The enzymes speci ied by hese genes a e espon-
sible o educ i e ans e o ammonium o 2-ke oglu a a e o
gene a e glu ama e (41, 42), which ac s as he majo ammo-
nium dono in he cell. The e a e wo pu a i e aspa a e ami-
no ans e ases, which ca alyze he e e sible ans e o he
amino g oup om glu ama e o oxaloace a e, ende ing aspa -
a e and 2-ke oglu a a e. This is a key enzyme as i links he
TCA cycle wi h he i s enzyme o he ec oines syn hesis pa h-
way (aspa okinase). C. salexigens has only one aspa okinase
ca alyzing he o ma ion o aspa yl phospha e, which is a com-
mon me abolic in e media e in he biosyn hesis o ec oines and
aspa a e amily o amino acids (8, 19). Fo a comple e desc ip-
ion o he in silico analysis, see supplemen al Table S1 and Figs.
S1–S4.
In Vi o Analysis
Ac i i ies o selec ed enzymes we e de e mined in i o. The
enzymes assayed belonged o ou main g oups as ollows: (i)
glucose/glucona e me abolism; (ii) TCA cycle; (iii) anaple o ic
and gluconeogenic eac ions, and (i ) ni ogen me abolism.
Enzyme ac i i ies we e de e mined in he mid-exponen ial
phase cul u es a di e en salini ies (Table 3).
FIGURE 3. Scheme o he cen al me abolism and syn hesis o ec oines in C. salexigens based on he anno a ed genome. Pa hways leading om glucose
o 6-P-glucona e (6PGln) a e p oposed on he compa ison o he in silico analysis o C. salexigens and P. pu ida. Abb e ia ions used a e as ollows: 2-KGlcn,
2-ke oglucona e; 2-Kglu, 2-ke oglu a a e; 6PGlcn, 6-phospho-D-glucona e; 6PKGlcn, 6-phospho-2-ke o-D-glucona e; AcCoA, ace yl-coenzyme A; AcP, ace yl
phospha e; Ala,L-alanine; Amm, ammonium; Asp,L-aspa a e; Asp-P,L-aspa yl phospha e; G3P, glyce aldehyde 3-phospha e; Glc,D- glucose; Glc6P,D-glucose
6-phospha e; Glcn,D-glucona e; Glcnlac,D-gluconolac one; Gln,L-glu amine; Gox, glyoxyla e; Glu,L-glu ama e; Ic ,D-isoci a e; KDGlcn6P, 2-ke o-3-deoxy-D-
glucona e-6-phospha e; Lac,D-lac a e; Lys,L- lysine; Mal, L-mala e; Me ,L-me hionine; NADA, N-
␥
-ace yl-L-2,4-diaminobu y a e; OAA, oxaloace a e; PEP, phos-
phoenolpy u a e; Py , py u a e; Suc, succina e; Th , L- h eonine; Acs, ace yl-coenzyme A syn he ase; Ac P, ace a e pe mease; AcyP, ace yl phospha e phospha-
ase; AlaAT, alanine amino ans e ase; Ald, alanine dehyd ogenase; AspAT, aspa a e amino ans e ase; AspK, aspa a e kinase; Cs, ci a e syn hase; Ec A,
diaminobu y a e ace yl ans e ase; Gad, glucona e dehyd ogenase; Gdhq, glucose dehyd ogenase; Gdh, glu ama e dehyd ogenase; Glk, glucokinase; Gls,
glu ama e syn hase; GnuK, gluconokinase; Icl, isoci a e lyase; KguD, 2-ke o-6-phosphoglucona e educ ase; KguK, 2-ke oglucona e kinase; Ldh, lac a e dehy-
d ogenase; Mae, malic enzyme; Mqo, mala e-quinone oxido educ ase; Ms, mala e syn hase; Oad, oxaloace a e deca boxylase; Pc, py u a e ca boxylase; Pdh,
py u a e dehyd ogenase; Pox, py u a e oxidase; Ppc, phosphoenolpy u a e ca boxylase; Pyk, py u a e kinase; Zw , glucose-6-phospha e dehyd ogenase.
Dashed a ows a e used o con e sions ha equi e mo e han one enzyma ic s ep.
Cen al Me abolism and Osmoadap a ion in C. salexigens
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Glucose Me abolic Enzymes, Glucose-6-phospha e Dehyd o-
genase and P k—Glucose-6-phospha e dehyd ogenase in e -
connec s he EM, ED, and pen ose phospha e pa hways (43)
and is conside ed as a majo ou e o NADPH p oduc ion o
biosyn hesis and edox homeos asis (Table 3).
Signi ican ly, we we e unable o de ec P k ac i i y abo e
backg ound in cell- ee ex ac s (Table 3 and supplemen al
Tables S1 and S2 and Figs. S1–S3). This ac along wi h he lack
o unequi ocal assignmen o P k sugges s ha , like Pseu-
domonads and some o he ae obic gene a (44), C. salexigens
uses he ED pa hway o glucose ca abolism, whe eas he ope -
a ion o unc ional glycolysis emains unce ain (Fig. 3).
TCA Cycle Enzymes, Cs and Icdh—In addi ion o hei
impo an ole o he supply o me abolic ene gy, TCA cycle
in e media es a e key building blocks o he syn hesis o bio-
mass and ec oines (Fig. 3). Two ac i i ies o he TCA cycle we e
de e mined, Cs and Icdh. Two Icdh-encoding genes a e anno-
a ed (Csal0525 and Csal1434), which di e in co ac o speci-
ici y; howe e , only NADP
⫹
-dependen Icdh ac i i y was
de ec ed. Rega dless o salini y, Icdh ac i i y was 8–15- old
highe han ha o Cs (Table 3).
Anabolic and Anaple o ic Pa hways Enzymes, Ppc, Pc, Isoci-
a e Lyase, and Malic Enzyme—Anaple o ic pa hways a e
essen ial o eplenish he OAA in he TCA cycle ha is wi h-
d awn o he p oduc ion o ec oines (Fig. 3). Bo h Ppc and Pc
ac i i ies we e de ec ed, he la e being 8–10- old highe han
he o me . In e es ingly, he Pc and Ppc ac i i ies measu ed in
cells g own a 2.5 MNaCl we e 2- old highe han obse ed a
low salini ies (Table 3).
The ac i i y o he anaple o ic enzymes isoci a e lyase
(isoci a e lyase and glyoxyla e shun ) and malic enzyme (malic
enzyme, gluconeogenesis) (45) was low compa ed wi h o he
ac i i ies analyzed. This is in ag eemen wi h wha has been
desc ibed in glucose-g own E. coli and Pseudomonas ae ugi-
nosa (46, 47).
Ni ogen Me abolism Enzymes, Aspa a e Amino ans e ase
and Glu ama e Dehyd ogenase—Glu ama e dehyd ogenase
(Gdh) along wi h glu amine syn he ase and glu ama e syn hase
a e he ou es o ammonium assimila ion in bac e ia. Bo h
NADH- and NADPH-dependen Gdh ac i i ies we e de ec ed
in C. salexigens ex ac s. Only he NADH-Gdh enzyme was
p edic ed om he genomic sequence o C. salexigens
(Csal1340) sugges ing ha i may no disc imina e be ween he
wo py idine nucleo ides. T ansaminases, ca alyzing he ans-
e o he amino g oup be ween amino acids, a e in ol ed in
amino acid syn hesis. Aspa a e amino ans e ase ac i i y was
high, which should no be su p ising conside ing ha his ac i -
i y mus accoun o he syn hesis o ec oines and all amino
acids om he aspa a e amily (Fig. 3).
Enzyme Ac i i ies and in Vi o Fluxes—Enzyme ac i i ies
de e mined in i o can be iewed as es ima es o lux h ough a
gi en ou e. In acellula ca bon luxes can be oughly es i-
ma ed om glucose up ake a es. When compa ed wi h Cs and
Pc ac i i ies, he ollowing is e iden : (i) Cs ac i i y was simila
a all h ee sal concen a ions, whe eas Pc ac i i y was highe
a 2.5 MNaCl (Table 3), and (ii) he glucose up ake a e was
5–30- old highe han Cs and Pc ac i i ies ( he di e ence being
highe a low salini y, supplemen al Table S3). Al hough his is
a ough app oxima ion, hese ac s a e in acco d wi h ou da a
on he accumula ion o ex acellula me aboli es (Table 2)
explaining why cells di e a signi ican pa o he me abolized
glucose o py u a e and ace a e o e low and sugges ing ha
he TCA cycle migh be limi ed by he low Cs ac i i y. In addi-
ion, he high Pc ac i i y indica ed ha OAA was ac i ely syn-
hesized om py u a e.
T acing Ec oines Labeling om Glucose
Finally, o assess he dis ibu ion o luxes o cen al me ab-
olism, he biosyn he ic pa hways o ec oines p oduc ion we e
aced by iso opic labeling wi h [1-
13
C]-, [6-
13
C]-, and [2-
13
C]g-
lucose a low and high salini y. All possible iso opome s de i ed
om hese p ecu so s ia he EM and ED pa hways, anaple osis
and he TCA cycle, we e p edic ed o py u a e, PEP, OAA,
ace yl-CoA, and ec oines, based on he anno a ed genome (sup-
plemen al Fig. S5 and Tables S3 and S4).
Ec oines Labeling in [1-
13
C]- and [6-
13
C]Glucose-g own
Cul u es—The analysis o he labeling o ec oines wi h [1-
13
C]-
and [6-
13
C]glucose p o ides a way o es ima e he EM o ED
lux a io. In cells g own wi h [1-
13
C]glucose, he COOH and
C6 o ec oines we e p edominan ly labeled, ega dless o he
sal concen a ion, and he labeling o he o he ca bons was
close o he na u al abundance o he
13
C iso ope (Fig. 4). The
labeling o COOH can be explained by use o he ED pa hway.
Su p isingly, C6 o ec oines con ained subs an ially highe
amoun o
13
C han would be p edic ed om na u al abun-
dance. We can accoun o his excess labeling o C6 o ec oines
by pos ula ing ha
13
CO
2
gene a ed by deca boxyla ion o
[1-
13
C]py u a e is einco po a ed e icien ly by he ca boxyla-
ion o ei he Pc o Ppc. The en ichmen o label a he COOH
o ec oines allowed us o es ima e ha o e 95% o glucose used
o ec oines syn hesis was me abolized h ough he ED pa hway
and Pc. Label sc ambling due o o he pa hways such as he
pen ose phospha e pa hways could explain mino label en ich-
men a o he posi ions. Also, he exis ence o a unc ional bu
mino glycolysis canno be i mly dismissed.
TABLE 3
Enzyme ac i i ies in c ude ex ac s o exponen ial phase o ba ch cul-
u es g own in glucose/M63 con aining 0.6, 0.75, and 2.5 MNaCl
All alues a e gi en in milliuni s䡠(mg p o ein)
⫺1
. Da a a e he a e ages o eigh
de e mina ions ( ou independen cul u es, assayed in duplica e). NM means no
measu ed. Zw is glucose-6-phospha e dehyd ogenase; Icl, isoci a e lyase; Mae,
malic enzyme; AspAT, aspa a e amino ans e ase.
Enzyme ac i i y
NaCl concen a ion
0.6 M0.75 M2.5 M
Glucona e me abolism
Zw 39.1 ⫾15.5 55.7 ⫾27.4 48.8 ⫾16.5
Glycolysis and TCA cycle enzymes
P k NM ⬍0.05 ⬍0.05
Cs 32.5 ⫾22.3 71.7 ⫾38.7 66.5 ⫾40.9
Icdh 462 ⫾189 645 ⫾295 554 ⫾183
Anaple o ic pa hway enzymes
Icl 1.27 ⫾0.69 2.30 ⫾1.24 1.93 ⫾0.70
Mae 5.69 ⫾2.86 7.99 ⫾6.83 5.87 ⫾2.18
Pc 92.2 ⫾19.1 70.6 ⫾8.2 167.0 ⫾28.0
Ppc NM 9.02 ⫾3.67 15.82 ⫾4.42
Ni ogen me abolic enzymes
NADPH-Gdh 22.3 ⫾7.60 73.7 ⫾31.4 16.4 ⫾9.0
NADH-Gdh 52.5 ⫾36.0 71.1 ⫾46.4 74.1 ⫾51.3
AspAT 95.2 ⫾18.0 174 ⫾93 78.5 ⫾16.5
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The esul s ob ained wi h [6-
13
C]glucose, which we e com-
plemen a y o hose ob ained wi h [1-
13
C]glucose, demon-
s a ed ha he inco po a ion o label in o ec oines om C1
and C6 o glucose is no equi alen (supplemen al Fig. S6). This
esul shows ha glucose ca abolism occu s h ough asymme -
ically labeled 3-ca bon me aboli es, con a y o wha would be
p edic ed o he EM pa hway. In addi ion, he mo e e icien
inco po a ion o
13
C label in o he glyce ol moie y o mem-
b ane phospholipids om [6-
13
C]glucose han om [1-
13
C]g-
lucose (supplemen al Figs. S7 and S8) is also in acco d wi h he
labeling o ec oines. Thus, hese da a demons a e ha C.
salexigens me abolizes glucose h ough he ED pa hway and,
oge he wi h he unce ain y o he exis ence o P k, sugges
ha he EM pa hway is no unc ional.
Ec oine Labeling in [2-
13
C]Glucose-g own Cul u es—Fo
[2-
13
C]glucose as ca bon sou ce, me abolism ia he EM pa h-
way would be p edic ed o yield PEP and py u a e ha a e bo h
50% labeled in hei C2. Me abolism ia he ED pa hway would
also gene a e [2-
13
C]py u a e bu would no p oduce any
[
13
C]PEP (supplemen al Fig. S5). The e o e, he me abolic a e
o he [2-
13
C]py u a e pool can be analyzed wi hou any u he
assump ions (Fig. 5). OAA could be syn hesized om py u a e/
PEP in C. salexigens by he ollowing ou es: (i) ca bons om
py u a e can en e he TCA cycle as ace yl-CoA p oduced by
Pdh, ende ing OAA a e one u n o he cycle (Fig. 5A); (ii)
py u a e and PEP can be ca boxyla ed o OAA by Pc o Ppc
(Fig. 5B), o (iii) by a combina ion o bo h ou es (Fig. 5C). F om
he spec a o ec oines, we can conclude ha he con ibu ion
o Ppc o he o al anaple o ic ac i i y o he cells is negligible (
supplemen al ma e ial). This would be in ag eemen wi h he
measu ed enzyme ac i i ies (Table 3).
Labeling o C6 and he ca boxylic g oup o ec oines inc ease
as a unc ion o he Pdh lux, whe eas labeling o C4 depends on
he Pc lux. The labeling o C2 o ec oines is he esul o he
inco po a ion by Ec A o he Pdh-p oduced ace yl-CoA. None
o he pa hway combina ions would yield ec oines labeled a he
me hyl g oup. These p edic ions i well wi h he co espond-
ing spec a, whe e he signal coming om he me hyl g oup is
he leas in ense and he mos in ense signals we e hose o C2
and C4 (Fig. 5E).
E ec o Salini y on Me abolic Fluxes, he Pc/Pdh, Cs/Ec A, and
Pc/Cs Flux Ra ios
To unde s and he unc ioning o cen al me abolic pa h-
ways in C. salexigens, he pa i ioning o py u a e and ace yl-
CoA can be desc ibed by he Pc/Pdh and Cs/Ec A a ios. The
Cs/Ec A lux a io desc ibes he ac ion o ace yl-CoA ha
en e s he TCA cycle e sus he ac ion ha is di ec ly inco -
po a ed in o ec oines. The Pc/Pdh lux a io indica es he ac-
ion o py u a e ha is ans o med o OAA e sus oxidized o
ace yl-CoA. Finally, he Pc/Cs a io allows compa ison o he
ac i i y o anaple osis and he TCA cycle and can be conside ed
as eadou o he biosyn he ic o ene ge ic needs o he cells
(Fig. 3).
The
13
C-labeling pa e n o ec oines syn hesized om
[2-
13
C]glucose can be used o quan i y ela i e luxes a hese
nodes (Table 4). Fo ha aim, he me abolic s eady s a e
hypo hesis was conside ed applicable o exponen ial cul u es
(and he e o e labeling a he speci ic posi ions o he ec oine
molecule is p opo ional o luxes). Peak a eas in
13
C NMR
spec a we e used o es ima e lux a ios, using simple algeb aic
equa ions (supplemen al ma e ial). The
13
C NMR spec a o
FIGURE 4.
13
C NMR spec a o in acellula ex ac s o [1-
13
C]glucose-g own cul u es. M63 minimal medium wi h 0.75 MNaCl (uppe spec um) and 2.5 M
NaCl (lowe spec um) was used. The signals co esponding o labeled ca boxylic ca bon (177 ppm o ec oine and 174–175 o hyd oxyec oine) and C6 (38–39
ppm o ec oine and 44 o hyd oxyec oine) a e shown. In he scheme, he expec ed a e o labeled ca bon when [1-
13
C]glucose is me abolized ia he
En ne -Doudo o pa hway is shown. I we assume ha he labeled C1 om py u a e is los as
13
CO
2
by deca boxyla ion a he le el o Pdh and inco po a ed
in o OAA by Pc, he p edic ed ec oines labeling pa e n would i he spec a ob ained. Rela i e labeling o each ca bon a om is indica ed by he colo scale a
igh . The abb e ia ions used a e as ollows: labeled compounds de ec ed: G, glu ama e; E, ec oine; H, hyd oxyec oine; GLC, glucose; PYR, py u a e; OAA,
oxaloace a e; AcCoA, ace yl-coenzyme A.
Cen al Me abolism and Osmoadap a ion in C. salexigens
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FIGURE 5. Inco po a ion o label om [2-
13
C]glucose in o ec oines.
13
C om [2-
13
C]py u a e (de i ed ia ei he he Embden-Meye ho o he En ne -
Doudo o pa hways), om [2-
13
C]phosphoenolpy u a e (made ia Embden-Meye ho ), o om unlabeled phosphoenolpy u a e (made ia En ne -Doudo-
o ) can be inco po a ed in o ec oines h ough he ollowing. A, oxaloace a e syn hesized in a single TCA cycle u n; B, oxaloace a e syn hesized by py u a e
ca boxylase o phosphoenolpy u a e ca boxylase (Pc/Ppc), o (C) oxaloace a e syn hesized by Pc/Ppc ollowed by a u n h ough he TCA cycle, which al e s i s
labeling pa e n (see he ex o de ails). D, scheme depic ing he ela ion o he py u a e and ace yl-CoA nodes wi h he ec oines biosyn hesis ou e in C.
salexigens.E,
13
C NMR spec a om in acellula ex ac s o [2-
13
C]glucose-g own cul u es. M63 minimal medium wi h 0.75 MNaCl (uppe spec um) and 2.5 M
NaCl (lowe spec um) was used. The signals co esponding o labeled ca boxyl ca bon, me hyl ca bon, C2, C4, and C6 o ec oine (E) and hyd oxyec oine (H) a e
shown. The signal co esponding o hyd oxyec oine ca boxyl ca bon o e laps wi h ha o C1 o glu ama e (indica ed as C1, G). No e ha o each pai o
chemical shi s co esponding o each ca bon, he a io o he ec oine/hyd oxyec oine signals is app oxima ely cons an . The h ee ec oine molecules in he
inse ep esen he iso opome dis ibu ions co esponding o a–c. Rela i e labeling o each ca bon a om is indica ed by he colo scale a igh o E. Whe e
applicable, he uppe hal o he co esponding ca bon posi ion ball depic s he expec ed labeling om [2-
13
C]py u a e/[2-
13
C]phosphoenolpy u a e, and he
lowe hal om unlabeled phosphoenolpy u a e. See supplemen al Table S4 and Fig. S5 and supplemen al ma e ial “De e mina ion o Me abolic Flux Ra ios”
o de ails.
Cen al Me abolism and Osmoadap a ion in C. salexigens
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