Download Cloning and expression of chromosomally and plasmid

FEMS MicrobiologyLetters 66 (1990) 29-34
29
Pubhshed by Elsevier
FEMSLE 03807
Cloning and expression of chromosomally and plasmid-encoded
glyceraldehyde-3-phosphate dehydrogenase genes
from the chemoautotroph Alcaligenes eutrophus
U t e WmdhiSvel a n d Botho Bowlen
]tL¢llllll file MJkeobtologle.Georg-Augusr-Umverstla!Gottmgen.Goltmgen.F R G
Received 14 August 1989
Revisionreceived17 AUgUSt1989
Key words" Gene duplication; cfx genes, gap gene; Megaplasmld priG1, lsoenzymes; Calvin cycle;
CO 2 fixation
1. SUMMARY
Hybndizauons using heterologous glycerald¢hyde-3-phosphate dehydrogenase ( G A P D H ) gene
probes suggested the existence of three G A P D H
genes in Alcahgenes eutrophus H16. Two of these,
located on the chromosome and the megaplasnud
priG1 of the orgamsm, respectively, mapped about
2.5 hlobase pairs (kb) downstream of the two
duplicated CO 2 ftxauon gene clusters (cfx genes)
They were identified as G A P D H genes (cfxGc and
cfxGp) by cloning and expression m Escherwhta
coh. These genes encode O A P D H tsoenzyrnes
functioning in the Calvin cycle. The durd gene
( g a p ) is chromosomaUy encoded but not hnked to
the cfx c cluster. Its product Is probably revolved
m heterotrophlc carbon metabolism.
Correspondenceto n Bowlen, |nstltut fur Mlkroblologte, Georg.August-UmversaatG6ttmgen, Gnsebachstrasse8. D-3400
Gonmgen, F R G
2. I N T R O D U C T I O N
The Calvin cycle is the metabohc route of CO:
assimilation in the facuhativdy autotrophlc hydrogan-oxtdtzmg bacterium Aleahgenes eutrophus
[1]. Except for its key enzymes nbulose-l,5-blsphosphate carhoxylase/oxygenase (RuBisCO) and
phosphonbulohnase (PRK) the cycle comprises
enzymes catalyzing reactions also involved m heterotropbac carbon metabohsm. Among these enzymes are fructose-l,6-/sedoheptulose-l,7-b~sphosphatase (FSBP) and glyceraldehyde-3-pbosphate dehydrogenase (GAPDH). A general question regarding the regulatory separatmn of the two
distinct modes of carbon metabohsm m facultatwely autotroph~c bacteria concerns the genetic
reformation for these enzymes.
Two apparently duphcated clusters of genes
(cfx genes) encoding Calvin cycle enzymes have
been found In the genome of Alealtgenes eutrophus
HI6. They are located on the chromosome and the
megaplasmld priG1 [2] that also cartaes the information for hydrogen oxidation [3]. So far, the
0378-1097/89/$03 50 © 1989 Federauon of European Microbiological Soc,eues
30
RuBisCO (cfxL, cfxS), PRK (cfxP) and FSBP
(cfxF) genes were identified as constituents of
both clusters [2,4]. Simultaneous expression of
these genes results m formatmn of Calvin cycle
tsoenzymes in the organism. The further analysis
of the efx regions lead to the detectmn of a
chromosomal GAPDH gene (cfxG¢) and a plasmad-encoded copy (cfxGp) each contained wtttun
the respecttve cfx cluster. This paper reports on
the locahzatton of the latter genes and their cloning and expressmn an Escher, chla coll. There ,s
ctrcumstantial evidence that .4. eutrophu~ possesses a tlurd GAPDH gene (des,gnated as gap)
whose product functions m glycolys,s/gluconeogenes~s.
3. MATERIALS AND METHODS
Resmctton enzymes, T4 DNA hgase and alkahne phosphatase were obtained from Gthco-
BRL (Eggenstem, F.R.G.), Bochnnger (Mannhe,m, F.R.G.) or Pharmacia LKB (Fre,hurg,
F.R.G.). The radiochcnucals and a rock-translation kit came from Amersham Buchler (Braunschwetg, F.ILG.).
Bacterial strains, phages and plasrmds used m
tlus study are listed in Table 1. A. eutrophus
strains were grown m Nument Broth (0.8%, w / v )
medmm at 30°C, E. cob XL1-Blue m LB-medmm
at 37°C and E coh DF221 also at 37°C either m
LB medium supplemented with 0.2% (w/v) glucose or in M9 mineral medmm [9] contaimng
succmate (0.4~, w / v ) and glycerol (0.1%, w/v).
For gene expression experiments E. coh was grown
m LB medium containing 50 /~g a m p i e d h n / m l
unUl reaching an optical density of 0.5 measured
at 550 nm, then 1 mM isopropyl4~-tluogalactos]de
(IPTG) was added and the cultures were incubated for additional 4 h.
Cells to be used for the preparauon of extracts
Table 1
Bacterial and phage strains and plasmldsused In this study
Strata or plasmld
Alcahgenes eutrophus
Eschertchla coil
H16
HF210 b
XLI-Blue
DF221
Pha~s
kAEC2
AEP2
Plasrmds
pUC19
pLOI312
pAEC4000
pAEC400I
pAEC4010
pAEP4000
pAEP400!
pAEp4010
Relevantcharactenstlcs a
Cfx, HOX:priG1
Cfx. Hox-, pHOl
Source or reference
DSM428. ATCC17699
B Fnednch
recA - (recAl #ac- endAl ~'rA96zht hsdRl7
supE44relAl {F' proAB laclq tacZAMI5 TnlO })
gap-2
[5l
15 7-kb incompletelydigested EcoRl fragment
from A eutrophuschromosomeinserted In ~L47
18-kb Incompletelydigested EcoRI fragment
from pHGI of A eutrophtts HI6 inserted i n ~L47
bin lacPOZ"
bin lacPOZ', pUC8 with an 1 7-kbrosen
carrying sap from ZymomonasmobJhs
3 7-kb EcoRl fragraent from gAEC2inserted m
pUCIg, cfxG~oriented m dtrecuon of lacPOZ"
3 7-kb EcoRI fragmentlikein pAEC4000but
cfxG~ onented opposite,o dlrecnon of lacPOZ"
2.5-kb Xhol/EcoRl fragmentfrom ?~AEC2inserted
In pUCt9, cfxG~ orientedm dffecuonof lacPOZ"
3 9-kb EcoRl fragmentfrom ~AEP2 inserted m
pUCIg, cfxGe onemed in d.reclion of lacPOZ'
3 9-kb Et'oRl fragmentlikein pAEP40tl0but
cfxG# orientedoppositeto drtectionof lacPOZ"
2 O-kb XhoI/EcoRI fragment from XAEP2inserted
m pUC19, cfxUporiented m dtrectlonof lacPOZ'
Cfx, ability1o fix CO2, tlox, abilityIn oxidizeH1.
b P|asrmd-free mutant derivedfrom A emrophtts H16
[6]
[2]
[2]
[7]
18]
This study
This study
This study
This study
This study
were harvested m the exponentml growth phase,
suspensed in 50 mM Tns-HCI, pH 7.5, containing
10 mM MgCI 2, 1 mM EDTA and 1 mM d*thiocrythntol at an optical denmty of about 125
measured at 436 nm and d~srupted by somhcalion. The supernatant resulting from a subsequent
centrifugatton of the homogenate at 25000 g at
4 0 C for 1 h was directly used for assays. The
actwity of G A P D H was deternuned as described
prevmusly [10] but using a pH of 8.5
Sodium dodecyl sulfate-polyacrylamtd¢ gel
eloctrophoresls (SDS-PAGE) was performed according to Laemmh [11].
Total bacterial D N A was isolated as detaded m
[12]. Estabhshed procedures [9] were employed to
isolate phage DNA. Large scale plasmid Isolation
was done by an alkahne SDS lysls procedure [13].
Transformants of E. cob XL1-Blue were tested for
plasrmd content by the rapid bothng method of
Holmes and Qulgley [14]. Digestions of D N A by
restricuon endonueleases and incubations in the
presence of alkahne phosphatase or T4 D N A hgase
were performed under the condtuons recommended by the suppher. Restnettou fragments
were isolated from agarose gels by the method of
Vogelstem et al. [15] using a kit (Geneclean, BIO
101, La Jolla, Ca., U.S.A.) For cloning experiments digested vector D N A was dephosphorylated. Ligated D N A was transformed into E col*
strums according to [16]. Transformants of E col,
XL1-Blue were selected on LB plates containing
50/x 8 amplelllm/ml. For complementauon analyXlS of E coh DF221 transformants the plates were
addmonally supplemented vath 1 raM IPTG.
Digested D N A was separated by agarose gel
electrophoresls and transferred to nylon membranes (GeneScreen Plus, Du Pont. Bad Homburg, F . R G . ) following the protocols of the
manufacturer. Southern hybndlzauon with 32p.
labelled probes were performed at 65 o C.
gene(s) m A eutrophus by means of heterolognas
hybridization probes appeared to be feasible. A
l.l-kb fragcaenl from pS198c encoding the cytosohc G A P D H of mustard (Smap~s alba) [19] and
a 1-kb P s t l / B a m H l fragment from pLOt312 carrymg the gap gone of Zymomonas mob~hs [8] were
used to probe restriction enzyme-digested D N A
from A. eutrophus H16 and its plasmld-eured
mutant HF210. The plant probe hybridized with a
chromosomal 14-kb EcoRl fragment (Fig. 1A, a, b)
that also showed weak homology to the bacterial
probe (Fig. ]B, c, d). However, no hybrid formation was observed between the mustard probe and
a chromosomal 3.7-kb EcoR.I or a pHGl-denved
A
B
kb
!
!
8
a
b
c
d
e
"~7
f
Fig 1 Lncahzatlon of GAPDH genes m the genome of A
4. RESULTS A N D DISCUSSION
4 1. Locahzaoon of GAPDH genes
Because of the strong structural conservation of
G A P D H s from orgamsms of different phylogenetic origin [17,18] localization of G A P D H
¢Utrophus HI6 by Southern hybridization using probes from
$mapls alba (A) or Z).momonasmobdts (B) labelled wah ~2p
DNA isolated from wdd-ty~e strata H16 (lanes a and c).
pHGl-cured mutanl HF210 (o and d) or recombinant phages
~AEC2 (¢) and ?~AEP2 (f) was digested with EcoR[ and
elcelrophoretlcally separated (10 [=$)m a 08% (w/v) agarose
gel Numbers indicate the sizes On kb) of hybridizing fragments
32
Table 2
Expressionof cfxG¢ and cfxGp from .4 euzroph~ measuredas
GAPDH actlvsty m transformants of E coh XLI-Bluecontmrung different plasrmds
B
~ ~'~' ~? ~
~
~
~'
F~g 2 Physicaland geneuc maps of the chromosomall~* (A)
and pHGl-encoded(B) cfx gene clusters of A eutrophusH16
The arrowsshow the ]ocauons and transcnptlonalorientations
of the genes Cleavagestiesfor resmctlonenzymes EcoRl (E),
8amHl (B) and Xhol (X) are 81ven Indices c and p refer to
chromosome and pHGI, respecevely
3 9-kb EcoRI fragment, both of wluch hybridized
strongly wtth the Z mobths probe (Fig. 1B, c, d).
Hybndtzatton of the latter probe with recombinant phages from a A. eutrophus gene bank (Ftg.
1B e, f) tdenttfied the two shorter EcoRI fragments as those located lmmedtately downstream
of the chromosomally and plasrmd-encoded cfxP
genes, respectively (Fig. 2). Probmg wtth the gapB
gene from the purple bacterium Rhodobacter
sphaeroldes [20] confirmed these hybndtzation
patterns (not shown) This su~ested the existence
of three GAPDH genes m A. eufrophus wtth two
of them, cfxG¢ and cfxGe, belongmg to the dupheared cfx clusters. The tlurd gene, gap, encoded
on the chromosovae (dtscussed in 4 3 ) is not hnked to the cfx genes
Plasnud
Specificactwltyof GAPDH
(U/rag extract protein)
3 92
40 36
4 02
42 20
23 62
3 86
8 90
pUC19
pAEC4000
pAEC400]
pAEC4010
pAEP4000
pAEP4001
pAEP4010
same transcripUonai orientation as the other cfx
genes.
These findings were substanttated and extended
by expression experiments designed to determine
the activity of the recombinant G A P D H isoenzymes and to detect the products of their genes.
The expt~esslons wece performed wtth transfor-
I,-
I
4 2 Subclonmg and expresston of the efxG genes m
E colt
The 3.7-kb and 3.9-kb EcoRI fragment and
their 2.5-kb and 2.0-kb XhoI/EcoRl subfragments, respecttvely (see Fig. 2), were cloned m E
colt using the expression vector pUC19 to prove
functmnality and identity of the cfxG genes. The
resuhmg hybrid plasmlds (see Table 1) were transformed into the gap mutant E. coh DF221 for
testing of phenotyptc trans complementation
Growth of transformants on LB agar would indicate expression of a GAPDH gene encoded on the
respective plasmid. All plasmids except pAECd00]
and pAEP4001 complemented the mutant, showing that the cfxG genes were mdeed funcUonal
and expressed under the control of the lae promoter of the vector. The genes apparently had the
-14
a
b
c
d
e
f
g
Fig. 3. SDS-PAGEof cell extracts (10 ~tgprotein) from transformants of E cob XLl-Blueharbonng differentple.smlds (a)
pUCI9, (b) pAEC4000, (c) pAEC400I, (d) pAEC4010, (e)
pAEP40~0, (I0 pAEP400I and (8) pAEP4010 The arrow head
points at the band of the putative recombinant GAPDH
subumt The electrophoresss was performed in a gel polymer.
Bed from 147o (w/v) acrylarmde Numbers mdeatc the sazes
0n kllodalton[kDa]) of molecularmass markers.
33
m a n t s of E. cob XL1-Blue smce m u t a n t D F 2 2 1
tended to loose the hybrid plasrmds. Extracts of
cells contaimng plasnuds with the cfxG genes
under control of the lac p r o m o t e r exhibtted twoto tenfold higher G A P D H acuvities t h a n those of
the reference strain h a r b o n n g p U C 1 9 ( T a b l e 2),
O p p o s i t e orientation of the genes did not cause an
increase o f enzyme activity. S D S - P A G E of these
extracts revealed a strong synthesis In the clones
with high G A P D H acuvity of a 35-kdodalton
polypeptide (Fig. 3). T h i s molecular mass correlates closely with that of subumts of other
G A P D H s [17], m a k i n g formatton of fuston proteins during heterologous expresston of cfxG unlikely.
4.3. Conclustens
T h e two cfxG genes of A. eutrophus H 1 6 do
not have promoters that are active m E colt.
However, their ribosome-bmding sites seem to be
recogmzed by the foreign host as in the case of the
o t h e r cfx genes [2]. T h e i r location relative to cfxP
resembles that of prkB and gapB m P~ sphaerotdes
[20]. It is possible that the cfxG genes of A
eutrophus lack own promoters and are p a r t of
large cfx operons whtch may comprise all genes
within each of the duplicated cfx clusters T r a n s poson T n S - m d u c e d mutants defective in C O 2 assimilation requtred a complete cluster including
cfxG for phenotyplc trans complementation (unpublished results). Thus, the phystologoeal role of
the cfxG-eueaded G A P D H lsoonzymes ts m the
C a l v i n cycle. Since these mutants with inactive
cfxG ,¢ere able to grow helerotrophically another
gene coding for a third G A P D H isoenzyme opera t m g in glycolysis and ghiconengenests must exast
m strata H ) 6 . T h i s gap cane is presumably located
on a chromosomal 14-kb EcoRl fragment (see Fig.
1).
ACKNOWLEDGEMENTS
W e are indebted to B. Bachmann, R. Cerff, T
Conway, D . G . Fraenkel, J.L. Gtbson, L.O. Ingrain
and F R T a h i t a for providing plasmads, probes or
strmns. Thts w o r k was supported by a g r a n t of the
Deutsche Forschangsgememschaft.
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