RESEARCH ARTICLE
Widesp ead occu ence
o non-phospho yla ing
glyce aldehyde-3-phospha e
dehyd ogenase among
g am-posi i e bac e ia
In oduc ion
Glyce aldehyde-3-phospha e dehyd ogenase (GAPDH) is an
enzyme in ol ed in cen al pa hways o ca bon me abolism.
The mos common o m o GAPDH is he NAD+-dependen
enzyme (EC 1.2.1.12) ound in all o ganisms so a s udied
and loca ed in he cy oplasm. This enzyme plays a ole in he
Embden-Meye ho pa hway no only in glycolysis bu also
in gluconeogenesis [8]. NADP+-dependen GAPDH (EC
1.2.1.13), loca ed in he chlo oplas s oma and he cyanobac-
e ial cy oplasm, is in ol ed in pho osyn he ic CO2assimila-
ion [3,5,33]. The non-phospho yla ing glyce aldehyde-3-
phospha e dehyd ogenase (GAPDHN; EC 1.2.1.9) is encod-
ed by he nuclea gene gapN and is ubiqui ous among pho o-
syn he ic euka yo es. While he enzyme is hough o me ab-
olize ioses expo ed om he chlo oplas , i s p ecise unc-
ion emains o be es ablished [25,30]. I does, howe e , ca -
alyze he oxida ion o glyce aldehyde-3-phospha e (G3P) o
3-phosphoglyce a e (3-PGA) wi h he educ ion o NADP+
o NADPH. No ino ganic phospha e is equi ed and he eac-
ion is i e e sible unde physiological condi ions. This is in
INTERNATIONAL MICROBIOLOGY (2005) 8:251-258
www.im.mic obios.o g
Summa y.The non-phospho yla ing glyce aldehyde 3-phospha e dehyd ogenase
(GAPDHN, NADP+-speci ic, EC 1.2.1.9) is p esen in g een euka yo es and some
S ep ococcus s ains. The p esen epo desc ibes he esul s o ac i i y and
immunoblo analyses, which we e used o gene a e he i s su ey o bac e ial
GAPDHN dis ibu ion in a numbe o Bacillus, S ep ococcus and Clos idium
s ains. Pu a i e gapN genes we e iden i ied a e PCR ampli ica ion o pa ial 700-bp
sequences using degene a e p ime s cons uc ed om highly conse ed p o ein
egions. Alignmen o he amino acid sequences o hese agmen s wi h hose o
known sequences om o he euka yo ic and p oka yo ic GAPDHNs, demons a -
ed he p esence o conse ed esidues in ol ed in ca aly ic ac i i y ha a e no
conse ed in aldehyde dehyd ogenases, a p o ein amily closely linked o
GAPDHNs. The esul s con i m ha he basic s uc u al ea u es o he membe s
o he GAPDHN amily ha e been conse ed h oughou e olu ion and ha no
iden i y exis s wi h phospho yla ing GAPDHs. Fu he mo e, phylogene ic ees
gene a ed om mul iple sequence alignmen s sugges ed a close ela ionship
be ween plan and bac e ial GAPDHN amilies. [In Mic obiol 2005; 8(4):251-
258]
Key wo ds:non-phospho yla ing glyce aldehyde-3-phospha e dehyd ogenase ·
Bacillus ·S ep ococcus ·Clos idium ·gapN genes
Recei ed 22 June 2005
Accep ed 19 Sep embe 2005
*Co esponding au ho s:
A. Se ano
Tel. +34-954489524. Fax +34-954460065
E-mail: [email p o ec ed]
A. Souk i
Tel. +212-22230672. Fax +212-22230674
E-mail: [email p o ec ed]
Abdelghani Idda 1
Fede ico Val e de2
Oma Assobhei3
Au elio Se ano2*
Abdelaziz Souk i1*
1Enzyma ic Enginee ing and
Molecula Gene ics Team,
Facul y o Sciences Aïn-Chock,
Uni e si y Hassan-II, Mâa i ,
Casablanca, Mo occo
2Ins i u e o Plan Biochemis y
and Pho osyn hesis (CSIC-
Uni . o Se ille), Se ille, Spain
3Lab. o Applied Mic obiology
and Bio echnology, Facul y o
Sciences, Uni . Chouaib Doukkali,
El Jadida, Mo occo
252 INT. MICROBIOL. Vol. 8, 2005
con as o he e e sibili y o NAD+- and NADP+-dependen
phospho yla ing GAPDH eac ions, which equi e ino ganic
phospha e o oxidize G3P in o diphosphoglyce ic acid [5,30].
GAPDHN was o iginally epo ed o be exclusi ely p esen in
g een euka yo es, and GAPDHN ac i i y was i s ound in he
cy osolic p o ein ac ion o lea issues, endospe m, and he
co yledons o plan s [21]. This was ollowed by he disco e y
o GAPDHN ac i i y in o he pho osyn he ic euka yo es, e.g.
in di e en algae [18,25]. Howe e , ea ly epo s o a non-
phospho yla ing GAPDH ac i i y in S ep ococcus mu ans [4]
we e con i med la e by molecula da a [2]. This s ain lacks
he wo oxida i e enzymes o he hexose monophospha e pa h-
way, glucose-6-phospha e dehyd ogenase and 6-phosphoglu-
cona e dehyd ogenase [4], and as a consequence mus use
al e na i e mechanisms, implica ing GAPDHN, o gene a e
NADPH o educ i e biosyn he ic eac ions. Recen ly, we
ha e cloned he gapN genes om Clos idium ace obu ylicum
and S ep ococcus pyogenes and exp essed hem in Esche i-
chia coli. The ecombinan GAPDHNs p oduced we e pu i ied
and hei physical and ca aly ic p ope ies in es iga ed [15,16].
The esul s showed ha hese genes e ec i ely encoded
GAPDHN p o eins wi h enzyma ic cha ac e is ics simila o
hose p e iously desc ibed.
The GAPDHN o highe plan s [10,11] and bac e ia [2]
consis s o a subuni o abou 490 amino acids. The ac i e
enzyme in plan s is a homo- e ame o abou 190 kDa, as
de e mined om bee , Chlamydomonas einha d ii, and
He ea b asiliensis [18,19,27]. The enzyme om S. mu ans,
C. ace obu ylicum, and S. pyogenes is a homo e ame wi h
subuni molecula masses o 51, 50, and 55 kDa, espec i ely
[15,16,22].
The amino acid sequences o GAPDHNs align well wi h
hose o aldehyde dehyd ogenases (ALDH), demons a ing
ha hey a e membe s o he la ge ALDH supe amily, sha -
ing an amino acid iden i y o 20–30%. Thus, GAPDHNs
clea ly di e om phospho yla ing GAPDHs bo h in p i-
ma y s uc u e and molecula mass [10,18,17]. Bac e ial and
plan enzymes o he GAPDHN amily ha e a much close
a ilia ion among each o he han wi h o he enzymes o he
ALDH supe amily. Fo example, S. mu ans GAPDHN
shows abou 50% amino acid iden i y wi h he enzyme o
pho osyn he ic euka yo es [11]. By con as , he a chaeon
Me hanococcus jannaschii has a non-phospho yla ing
GAPDH wi h e edoxin-dependen ac i i y, bu he
sequence o he enzyme is dis inc ly sepa a e om ha o
NADP+-dependen GAPDHNs [11]. To cla i y he dis ibu-
ion o GAPDHN in bac e ia, we su eyed he occu ence
and ac i i y o he enzyme in se e al bac e ial s ains, apply-
ing a molecula gene ic app oach o ga he mo e in o ma ion
on he enzyme’s dis ibu ion.
Ma e ial and me hods
S ains and cul u e condi ions. Clos idium ace obu ylicum
ATCC 824 and ATCC 859, C. pe ingens ATCC 13124, C. pas eu ianum
ATCC 6013, C. di icile ATCC 11011 and C. spo ogenes CIP 79.39 s ains
we e g own in yp icase-yeas -ex ac -glucose (TYA) b o h [26] a 37ºC in
an anoxic chambe wi hin a ni ogen a mosphe e. S ep ococcus pyogenes
[16], S. agalac iae ATCC 13813 and isola ed S ep ococcus sp. s ains we e
g own in Todd Hewi b o h (THY) medium con aining 0.2% (w/ ) yeas
ex ac [1]. Bacillus mega e ium ATCC 14945, B. sub ilis ATCC 6633, B.
hu ingiensis ATCC 10792, S aphylococcus au eus ATCC 25923, Myco-
bac e ium ube culosis ATCC 27294, Pseudomonas ae uginosa ATCC
9027, Bac e oides agilis ATCC 25285, En e ococcus aecium CIP 54.32,
and E. hi ae ATCC 10541, and isola ed Bacillus sp., B. licheni o mis, and B.
ce eus [14], we e g own a 37ºC in Lu ia-B o h (LB) medium [29].
Neisse ia meningi idis M13 was g own a 37ºC on GCB medium (Di co
Labo a o ies, USA) [20]. Lac obacillus b e is ATCC 14869, L. pa acasei
ATCC 25598, L. plan a um ATCC 8014, and L. lac is CNRZ 548 we e
g own a 25°C in MRS medium [7].
Cell- ee ex ac p epa a ion.Liquid cul u e cells we e ha es ed
by cen i uga ion a 8,000 × g o 15 min a 4ºC. Cell pelle s we e washed
wice in 25 mM T is-HCl (pH 7.5) and esuspended in he same bu e sup-
plemen ed wi h 2 mM di hio h ei ol (DTT), 1 mM phenylme hylsul onyl
luo ide (PMSF), and 10% ( / ) glyce ol. Cells we e hen dis up ed by ul a-
sonic ea men in a chilled wa e ba h using a B anson 25U soni ie a medi-
um s eng h. The esul ing suspension was cen i uged a 20,000 × g o 20
min o ob ain he cell- ee ex ac .
Enzyme assays
.
GAPDHN ac i i y was measu ed as desc ibed else-
whe e [34]. The eac ion was s a ed by he addi ion o 10 µg o cell- ee
ex ac o an assay mix u e con aining 50 mM T icine bu e (pH 8.5), 1 mM
NADP+, and 1 mM D-glyce aldehyde-3-phospha e a 25ºC. Abso bance a
340 nm was ollowed in a spec opho ome e (model 6405, Jenway,
Dunmow, UK). Phospho yla ing NAD+-dependen GAPDH ac i i y was
measu ed using he same p ocedu e bu employing NAD+(1 mM) and ino -
ganic phospha e o a sena e (10 mM) in he eac ion solu ion.
P o ein immunode ec ion
.
Immunoblo assays o p o ein samples
we e ca ied ou a e SDS-PAGE [12% (w/ ) polyac ylamide slab gels] as
desc ibed in [15] o C.ace obu ylicum.
PCR me hodology and DNA sequencing. Ampli ica ion o ca. 0.7-kb ag-
men s o gapN genes om di e se bac e ial genomic DNA samples was ca -
ied ou by PCR using degene a e p ime s om wo highly conse ed
egions a he N- e minal and C- e minal ends o GAPDHN p o eins
(NPCO1: 5′-C(T)TA(G)GCT(CAG)ATT(CA)T(A)C(G)T(C)CCT(CAG)TT
T(C)AAT(C)-3′, and NPCO2: 5′-CCT(CAG)GGT(CAG)TTT(C)CCT(CA
G)GAA(G)GAA(G)TGG-3′). Ampli ica ion condi ions we e: cycle 1, 92°C
o 2 min; cycles 2–36, 92°C o 1 min, 45°C o 1 min, and 72°C o 1 min;
cycle 37, 72°C o 30 min. Ch omosomal DNA was isola ed using a Wiza d
Ki (P omega, Madison, USA). The ampli ied eac ion was isualized on
0.8 % (w/ ) aga ose gels wi h he addi ion o e hidium b omide acco ding o
[29]. PCR-ampli ied DNA agmen s we e pu i ied by selec i e adso p-
ion/deso p ion on glass beads (Gene Clean, Bio101, La Jolla, CA, USA).
Sequence analysis was ca ied ou employing he DNA S ide p og am ( e -
sion 1.2 o Macin osh).
DNA alignmen and phylogene ic analyses. Mul iple sequence
alignmen o GAPDHN p o ein egions co esponding o he PCR-ampli ied
DNA agmen s o he bac e ia s udied we e done wi h he Clus al X .1.8
p og am [32]. Using his alignmen , phylogene ic ees we e cons uc ed
employing he dis ance (neighbo -joining, Kimu a dis ance calcula ions),
IDDAR ET AL.
253
INT. MICROBIOL. Vol. 8, 2005
maximum likelihood, and maximum pa simony me hods and using he p o-
g ams Clus al X .1.8, T ee-Puzzle .5.0 [31] and P o pa s .3.573c
(PHYLIP package .3.5c w1993x Felsens ein, J., Dep . o Gene ics, Uni . o
Washing on, Sea le, USA), espec i ely. Boo s ap analyses ( alues p esen -
ed on a pe cen age basis) we e compu ed wi h 1000 eplica es o he dis-
ance and maximum pa simony ees; o maximum likelihood analysis, es i-
ma ions o suppo we e assigned o each in e nal b anch using he qua e
puzzling algo i hm [31]. Published amino acid sequences o GAPDHNs
used in his wo k come om bac e ia (Bacillus an h acis, accession numbe
AAP24851; Bacillus halodu ans, E83929; S ep ococcus mu ans,
NP721104; S ep ococcus pneumoniae TIGR4, NP345590; S ep ococcus
pneumoniae R6, NP358622; Mycoplasma cap icolum, CAA 83756;
U eaplasma u ealy icum, AAF30771), highe plan s (Pisum sa i um,
P81406; Nico iana plumbagini olia, P93338; Zea mays, Q43272), and a
mic oalga (Scenedesmus acuola us, CAC81014). Fe edoxin-dependen
GAPDHN sequences o a chaea (Py ococcus u iosus, NP578193 and
Me hanococcus jannaschii, NP248149) we e also used. Bac e ial GAPDH
encoded by he Esche ichia coli gap1 gene (accession numbe P06977) was
used as ou -g oup. PCR-ampli ied pa ial gapN sequences om bac e ia
we e submi ed o EMBL/GeneBank da abases and assigned accession num-
be s as ollows: AJ880320 (Clos idium ace obu ylicum ATCC859),
AJ880322 (C. pas eu ianum), AJ880325 (C. di icile), AJ880321 (C. pe -
ingens), AJ880323 (C. spo ogenes), AJ8800317 (Bacillus sp.), AJ880318
(B. ce eus), AJ880319 (B. licheni o mis), AJ880324 (B. hu ingiensis),
AJ880316 (S ep ococcus sp.), AJ880315 (S. agalac iae), and AJ880326 (S.
pyogenes).
Resul s and Discussion
GAPDHN ac i i y and immunoblo analyses.
Table 1 shows he enzyma ic ac i i ies (U/mg) o phospho y-
la ing NAD+-dependen and non-phospho yla ing NADP+-
dependen glyce aldehyde-3-phospha e dehyd ogenase
(GAPDH and GAPDHN) in he s ains s udied. Phospho-
yla ing NAD+-dependen GAPDH was p esen uni e sally
and showed high speci ic ac i i y alues, in he ange o
0.2–2.5 U/mg, in all bac e ial s ains examined. By con as ,
GAPDHN ac i i y (speci ic ac i i y 0.01–0.1 U/mg) was
ound only in a numbe o g am-posi i e s ains o he gen-
NON-PHOSPHORYLATING GAPDHN
Table 1. Phospho yla ing NAD+-dependen (GAPDH) and non-phospho yla ing NADP+-dependen
glyce aldehyde-3-phopha e dehyd ogenase (GAPDHN) ac i i ies in di e en bac e ial s ains
Bac e ia GAPDH ac i i y (U/mg) GAPDHN ac i i y (U/mg)
Clos idium ace obu ylicum ATCC824 1.010 0.013
Clos idium pe ingens 0.192 0.011
Clos idium pas eu ianum 0.190 0.060
Clos idium di icile 0.265 0.040
Clos idium spo ogenes 2.300 0.037
S ep ococcus pyogenes 2.510 0.017
S ep ococcus agalac iae 0.282 0.090
S ep ococcus sp.0.400 0.102
Bacillus licheni o mis 0.188 0.099
Bacillus ce eus 0.200 0.040
Bacillus hu ingiensis 0.312 0.010
Bacillus sp. 1.000 0.038
S aphylococcus au eus 1.229 nd*
Bacillus mega e ium 1.420 nd
Bacillus sub ilis 1.030 nd
Bac e oides agilis 0.500 nd
Neisse ia meningi idis 0.750 nd
En e ococcus aecium 1.006 nd
En e ococcus hi ae 2.240 nd
Lac obacillus b e is 0.543 nd
Lac obacillus pa acasei 0.465 nd
Lac obacillus plan a um 0.733 nd
Lac ococcus lac is 0.312 nd
Mycobac e ium ube culosis 0.900 nd
Pseudomonas ae uginosa 1.000 nd
*nd, no de ec ed.
254 INT. MICROBIOL. Vol. 8, 2005
e a Clos idium, S ep ococcus, and Bacillus. Su p isingly, de-
spi e some cases o close e olu iona y linkage o hose bac e ia,
ac i i y was absen om B. mega e ium, B. sub ilis, E. aecium,
E. hi ae, S. au eus, L. b e is, L. pa acasei, L. plan a um, L.
lac is, M. ube culosis, and he g am-nega i e bac e ia N.
meningi idis,Ba. agilis, and P. ae uginosa.
Figu e 1 shows he immunoblo con aining single-p o ein
bands o ca. 48–54 kDa, co esponding o he GAPDHN sub-
uni s, in soluble p o ein ex ac s om he species men ioned
abo e ha ha e non-phospho yla ing dehyd ogenase ac i i y
Simila bands we e de ec ed by immunoblo s and using he
same an ibodies in cell ex ac s om o he bac e ia, includ-
ing Clos idium pas eu ianum and C. spo ogenes (da a no
shown). The obse ed c oss- eac ion sugges s common epi-
opes be ween he C. ace obu ylicum p o ein and he o he
bac e ial GAPDHNs. As expec ed, no bands we e immuno-
de ec ed in cell ex ac s o hose bac e ia in which ac i i y
had no been eco ded.
PCR and sequence analysis o bac e ial
gapN
genes. The bands esul ing om sepa a ing he PCR p od-
uc s on aga ose gel a e shown in Fig. 2. A e ampli ica ion
o he ca. 0.7-kb single DNA agmen s, hese bands we e
excised om he gel, sequenced, and hei deduced amino
acid composi ion analyzed. All o he amino acid sequences
showed good iden i y wi h ha om he gapN agmen o S.
mu ans. In ag eemen wi h biochemical and immunochemi-
cal da a, no band was ampli ied in he cases o B. mega e-
ium, B. sub ilis, Ba. agilis, S. au eus, N. meningi idis, E.
aecium, E. hi ae, L. b e is, L. pa acasei, L. plan a um, L.
lac is, M. ube culosis, and P. ae uginosa.
Figu e 3 shows he alignmen be ween he app oxima ely
240 amino acids co esponding o he ampli ied 700-bp ag-
men s and he deduced amino acid sequences o he co e-
sponding GapN agmen s om highe plan s (P. sa i um, N.
plumbagini olia, Z. mays) and algae (S. acuola us). The la -
e we e also compa ed wi h o he deduced amino acid
sequences o bac e ial GAPDHNs in da abases (B. an h acis,
B. halodu ans,S. mu ans, M. cap icolum, U. u ealy icum).
As expec ed, he non-phospho yla ing GAPDHN showed
no signi ican iden i y ma ch wi h phospho yla ing GAPDHs.
Bo h non-phospho yla ing GAPDH and phospho yla ing
GAPDHs showed high speci ici y owa ds D-glyce aldehyde-
3-phospha e [13,18], which sugges ed ha subs a e speci-
ici y o he wo GAPDH o ms eme ged by con e gen e o-
lu ion along independen lines [10]. Sequences o he
a chaeal e edoxin-dependen GAPDHN, which equi es a
hea y-me al co ac o and is oxygen sensi i e, we e dis inc -
ly di e en om ha o any o he NADP+-dependen
GAPDHN (no shown).
The 12 deduced pa ial bac e ial GAPDHN sequences
de e mined in his s udy we e compa ed using he CLUSTAL
X ( . 1.8) p og am wi h o he published amino acid sequen-
ces o euka yo ic and p oka yo ic o igin. The esul s showed
signi ican simila i ies be ween he a ious p oka yo e and
euka yo e sou ces (Fig. 3). In addi ion o he conse ed hep-
apep ide Se -Gly-Glu-A g-Cys-Th -Ala ( esidues 294–300,
ollowing pea GAPDHN numbe ing [10], including esidues
A g-297, necessa y o phospha e g oup binding o he sub-
s a e, and Cys-298, in ol ed in ca aly ic hioes e o ma-
ion), o he esidues in ol ed in enzyme ac i i y a e s ic ly
conse ed in all GAPDHN p o eins. These a e he 191–192
dipep ides Lys-P o, and he hexapep ide Glu-Leu-Gly-Gly-
Lys-Asp a posi ion 264–269. The la e includes he ac i e
si e Glu-264, in ol ed in deacyla ion h ough ac i a ion and
o ien a ion o he a acking wa e molecule [22,23,24] (Fig.
3). Thus, he basic s uc u al ea u es o he membe s o he
GAPDHN amily ha e been conse ed o e e olu ion. The
highly conse ed Gly-249 and Gly-295 a e also p esen in he
aldehyde dehyd ogenase supe amily [10], showing ha
IDDAR ET AL.
In . Mic obiol.
Fig. 1. Immunode ec ion o non-phospho yla ing glyce aldehyde 3-phos-
pha e dehyd ogenase (GAPDHN) in c ude ex ac s o a ious bac e ia.
Lane 1 Clos idium ace obu ylicum ATCC 824, lane 2 C. di icile,lane 3
S ep ococcus agalac iae,lane 4 S ep ococcus sp., lane 5 C. spo ogenes,
lane 6 S. pyogenes,lane 7 C. pe ingens,lane 8 Bacillus ce eus,lane 9 B.
hu ingiensis,lane 10 B. licheni o mis. App oxima ely 50 µg o c ude
ex ac s we e loaded pe lane. Appa en molecula masses a e indica ed on
he le .
In . Mic obiol.
Fig. 2. PCR ampli ica ion o he 700-bp agmen om he gapN gene,
employing ch omosomal DNA o di e se bac e ia. Lane 1 Clos idium ace-
obu ylicum ATCC859, lane 2 C. pas eu ianum,lane 3 C. di icile,lane 4 C.
pe ingens,lane 5 C. spo ogenes,lane 6 B. hu ingiensis,lane 7 Neisse ia
meningi idis,lane 8 Lac obacillus b e is,lane 9 B. ce eus,lane 10 S ep-
ococcus pyogenes,lane 11 S ep ococcus sp. Bands we e isualized on
0.8% (w/ ) aga ose gels in he p esence o e hidium b omide. HindIII-
es ic ed lambda DNA was used as he molecula size ma ke (M). The
a ow indica es he ampli ied gapN agmen s.
255
INT. MICROBIOL. Vol. 8, 2005
NON-PHOSPHORYLATING GAPDHN
In . Mic obiol.
Fig. 3. Mul iple sequence
alignmen using he
CLUSTAL X .1.8 p o-
g am o he pa ial
GAPDHN sequences om
pho osyn he ic euka yo es
and bac e ia. Sequences
ob ained in his wo k as
well as published amino
acid sequences o bac e ial
and plan sou ces, as des-
c ibed in Ma e ials and
me hods, we e included.
The conse ed mo i es
including he ca aly ic im-
po an esidues Glu 264
and Cys 298 (showed in
bold) a e unde lined. The
degene a e p ime s used in
PCR expe imen s a e indi-
ca ed by a ows.

256 INT. MICROBIOL. Vol. 8, 2005 IDDAR ET AL.
GAPDHNs and ALDHs p obably sha ed a common ances o .
Ne e heless, he numbe o esidues in which ALDHs di e
om GAPDHN in he aligned sequences p o ides e idence
ha hey ac ually belong o di e en enzyme amilies.
Phylogene ic analysis o GAPDHN sequences.
The phylogene ic ee cons uc ed using he abo e-desc ibed
mul iple sequence alignmen and he dis ance (neighbo -join-
ing) me hod [28] is shown in Fig. 4. Maximum likelihood
and pa simony me hods we e also used, gi ing esul s (no
shown) e y simila o hose in Fig. 4. E. coli GAPDH1 p o-
ein was used as ou -g oup. The ees showed an analogous
phylogene ic ela ionship among he GAPDHNs o he
euka yo ic and p oka yo ic g oups. These g oups a e di e -
en om he a chaeal e edoxin-dependen GAPDHN,
which p obably di e ged ou i s , be o e euka yo ic and bac-
e ial GAPDHN sepa a ion. Howe e , no e ha hese esul s
depic only he molecula phylogeny o he GAPDHN p o-
ein and do no necessa ily ep esen phylogene ic ela ion-
ships be ween species. A pa aphyle ic ela ionship is
obse ed be ween bac e ial GAPDHN sequences ha appea
in h ee clus e s (S ep ococcaceae, Clos idia, and
Bacillaceae) oge he wi h he plan g oup, and a sepa a e,
ea ly-b anching g oup o mycoplasm sequences (U ea-
In . Mic obiol.
Fig. 4. Dis ance phylogene ic ee o he
bac e ial GAPDHN sequences deduced
om he gapN agmen s, as desc ibed in
Ma e ials and me hods. Sequences o
Clos idium ace obu ylicum ATCC 859,
C. pe ingens,C. pas eu ianum,C. di i-
cile,C. spo ogenes,S ep ococcus pyo-
genes,S. agalac iae,S ep ococcus sp.,
Bacillus licheni o mis,B. ce eus,Bacillus
sp., we e ob ained in his wo k (in bold).
257
INT. MICROBIOL. Vol. 8, 2005
plasma u ealy icum and Mycoplasma cap icolum). This sug-
ges s ha GAPDHNs o Mycoplasma aceae sepa a ed ea ly
in e olu ion. Indeed, mycoplasms include some o he small-
es p oka yo ic genomes (600 kb, abou 500 genes), and he
sequences o many genes a e e y di e gen when compa ed
o homologous sequences o o he bac e ia [9]. These phylo-
gene ic ela ionships may be due o ho izon al gene ans e s
and enzyme unc ional subs i u ions, such as hose desc ibed
o some GAPDHs [6,8,12].
In his wo k, we demons a ed ha he gapN gene is p es-
en in a ious g am-posi i e bac e ia wi h a cha ac e is ic
low G + C con en , including Bacillaceae, S ep ococcaceae,
and Clos idiaceae. A possible explana ion o his dis ibu-
ion among some g am-posi i e bac e ia and o he absolu e
absence o gapN in g am-nega i e bac e ia could be an ea ly
di e gence in basic me abolic enzymes. Ne e heless, a
wide su ey o his p o ein among o he mic oo ganisms
would p o ide be e knowledge o i s dis ibu ion and, espe-
cially, i s ela ion wi h GAPDHs.
Acknowledgemen s. This wo k was suppo ed by AECI (Spain) and
Collabo a i e G an s om he Andalusian Go e nmen (Conseje ía de
P esidencia, Jun a de Andalucía, Ayuda de Coope ación al Desa ollo en el
Ambi o Uni e si a io no. 52/02, 54/04, and PAI CVI-261 g oup) – Minis é e
d’Educa ion e de la Reche che Scien i ique and PARS (Mo occo). FV was
suppo ed by an EU Ma ie Cu ie ein eg a ion p ojec no. 505303.
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NON-PHOSPHORYLATING GAPDHN
Amplia dis ibución de la glice aldehído-3-
os a o deshid ogenasa no- os o ilan e en e
las bac e ias g am-posi i as
Resumen. La glice aldehído-3- os a o deshid ogenasa no- os o ilan e
(GAPDHN, NADP+-especí ica, EC 1.2.1.9) es á p esen e en o ganismos
euca io as o osin é icos y en algunas cepas de S ep ococcus yClos idium.
En es e abajo se p esen an los esul ados de los análisis de ac i idad e
inmuno ans e encia, que se u iliza on pa a la p ime a p ospección de la dis-
ibución de GAPDHN bac e iana en di e sas cepas de Bacillus,S ep-
ococcus yClos idium. Se han iden i icado genes pu a i os gapN median e
ampli icación po PCR de secuencias pa ciales de 700 bp u ilizando ceba-
do es degene ados cons uidos a pa i de egiones p o eínicas muy conse -
adas. Las secuencias de aminoácidos de es os agmen os se alinea on con
las de o as secuencias conocidas de GAPDHN euca ió icas y p oca ió icas,
lo que demues a la p esencia de esiduos conse ados que pa icipan en la
ac i idad ca alí ica y que no se han conse ado en las aldehído deshid oge-
nasas, una amilia de p o eínas es echamen e elacionadas con las
GAPDHN. Los esul ados con i man que las ca ac e ís icas es uc u ales
básicas de los miemb os de la amilia GAPDHN se han conse ado du an e
la e olución y que no exis e iden idad con las GAPDH os o ilan es.
Además, los á boles ilogené icos gene ados a pa i de alineaciones de
secuencia múl iples sugie en una es echa elación en e las amilias
GAPDHN en plan as y bac e ias. [In Mic obiol 2005; 8(4):251-258]
Palab as cla e: glice aldehído-3- os a o deshid ogenasa no os o ilan e
·Bacillus ·S ep ococcus ·Clos idium · genes gapN
Ampla dis ibuição da glice aldeído-3- os a o
desid ogenase não os o ilado a en e as
bac é ias g am-posi i as
Resumo.Indicou-se a p esença da glice aldeído-3- os a o desid ogenase
não os o ilado a (GAPDHN, NADP+-especí ica, EC 1.2.1.9) em o ganis-
mos euca io as o ossin é icos e em algumas cepas de S ep ococcus eClos-
idium. Nes e abalho ap esen a-se os esul ados da a i idade en imuno-
ans e ência, usados pa a a p imei a p ospecção da dis ibuição da GAPDHN
bac e iana em di e sas cepas de Bacillus,S ep ococcus eClos idium. Se
iden i ica am genes pu a i os gapN median e ampli icação po PCR de
seqüências pa ciais de 700 bp u ilizando iniciado es degene ados cons uí-
dos a pa i de egiões p o eicas al amen e conse adas. As seqüências de
aminoácidos des es agmen os se alinha am com as de ou as seqüências
desconhecidas de GAPDHNs euca ió icas e p oca ió icas, o que demons a
a p esença de esíduos conse ados que pa icipam da a i idade ca alí ica
que não es ão conse ados nas aldeído desid ogenases, uma amília de p o-
eínas es ei amen e elacionados com as GAPDHN. Es e abalho con i ma
que as ca ac e ís icas es u u ais básicas dos memb os da amília GAPDHN
se conse a am du an e a e olução e que não exis e iden idade com as
GAPDH os o ilan es. Além disso, as á o es ilogené icas ge adas a pa i
de alinhamen os de seqüência múl iplas indicam uma es ei a elação en e
as amílias de GAPDHN de plan as e bac é ias. [In Mic obiol 2005;
8(4):251-258]
Pala as cha e:glice aldeído-3- os a o desid ogenase não os o ilado a
·Bacillus ·S ep ococcus ·Clos idium · genes gapN
258 INT. MICROBIOL. Vol. 8, 2005
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