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JOURNAL OF BACrERIOLOGY, Nov. 1993, P. 7495-7499 Vol. 175, No. 22


0021-9193/93/227495-05$02.00/0
Copyright C 1993, American Society for Microbiology

Cloning, Sequencing, and Characterization of the Gene


Encoding the Class I Fructose-1,6-Bisphosphate
Aldolase of Staphylococcus carnosus
CLAUDIA WITKE AND FRIEDRICH GOTZ*
Mikrobielle Genetik, Universitat Tubingen, Waldhauser Strasse 70/8, 72076 Tubingen, Germany
Received 26 May 1993/Accepted 13 September 1993

fda from Staphylococcus carnosus TM300, encoding the class I fructose-1,6-bisphosphate aldolase, was cloned
in Escherichia coli and sequenced. The 888-nucleotide open reading frame encoding a protein with an Mr of
32,855 had an E. coli-like promoter sequence. Plasmids containing fda complemented E. coli NP315 (Fda-).
Expression offda in S. carnosus led to a six- to eightfold increase in aldolase production and activity; low levels
of glucose in the growth medium stimulated activity.

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Fructose-1,6-bisphosphate (FBP) aldolase (EC 4.1.2.13) is a been already initiated, it is necessary to know the protein
glycolytic enzyme which catalyzes the reversible cleavage of sequence and to have the cloned gene available to determine
fructose-1,6-bisphosphate to form dihydroxyacetone phos- the refolding pathway by using specific mutations. Here we
phate and glyceraldehyde 3-phosphate. There are two classes report the isolation and DNA sequence of the class I aldolase
of FBP aldolases. Class I aldolases, typical of higher eucaryotes gene from S. carnosus and its expression in the donor strain.
such as animals and plants, consist of tetramers with a total Chromosomal location of fda. The aldolase of S. carnosus
molecular weight of about 160,000 (16). Class I aldolases form has been purified (5). On the basis of the N-terminal sequence
an intermediate with their substrate through a Schiff base by
condensation of the carbonyl group with the s-amino group of
a lysyl residue in the active center. This complex can be fixed by
reduction with NaBH4, leading to an irreversible inactivation A.
of the enzyme (16).
In contrast, FBP aldolases of most bacteria are class II
aldolases. They do not form a Schiff base intermediate; they
require a divalent cation, such as Ca2+, Fe2+, or Zn2+, to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
stabilize the carbanion intermediate of the enzyme-substrate K * h00rj<S40
complex (15); and they can be inhibited by EDTA (16). Since
it was believed that class I aldolases were restricted to higher
eucaryotic organisms, it was a surprise to find them in Pepto-
coccus aerogenes (20), Lactobacillus casei (21), Escherichia coli
(33), and most staphylococcal species (12).
The class I aldolase of Staphylococcus aureus has been
studied in detail. The enzyme is unusually heat resistant and
extremely acid and base stable (11). While the rabbit muscle
aldolase is active as a tetramer, the 33-kDa S. aureus aldolase B.
is active as a monomer. The kinetics of thermally and guani-
dinium chloride-induced unfolding and refolding of this aldo-
lase are complex and comprise at least one fast and two slow
reactions (25). Recently it was found that the class I aldolase of Clal
the staphylococcal cloning host Staphylococcus carnosus (10) is
also unusually temperature and pH stable (4, 5). Furthermore, PstI HindIII EcoRI Pstl
the S. carnosus aldolase reacts with a wide range of aliphatic ClaI EcoRI EcoRV HindIlI XbaI
PvuIII
aldehydes and is much more stable than the rabbit aldolase.
Because of its substrate range and stability, the S. carnosus
I I I

aldolase is the enzyme of choice for large-scale synthesis of


glycosidase inhibitors (36), 6-desoxy-fructose-1-phosphate fda
(35), C8 and Cg sugars (2), and similar compounds. FIG. 1. (A) Hybridization of Aldol to S. carnosus TM300 genomic
From the biophysical point of view, the staphylococcal class DNA. Autoradiograph of the Southern blot hybridized with purified
I aldolase could become a model enzyme to study unfolding and 32P-labeled Aldol. Chromosomal DNA was digested with the
and refolding kinetics. To continue those studies which have following restriction endonucleases. Lanes: 1, ClaI; 2, ClaI-HindIII; 3,
ClaI-PstI; 4, ClaI-BamHI; 5, HindIII; 6, HindIII-PstI; 7, HindIII-
BamHI; 8, BamHI-PstI; 9, HindIII-ClaI-PstI; 10, HindIII-ClaI-EcoRI;
11, PvuII; 12, PvuII-EcoRI; 13, PvuII-HindIII; 14, PvuII-ClaI; 15,
PvuII-PstI; 16, PvuII-XbaI; 17, ClaI-XbaI; 18, PstI-XbaI. (B) Location
*
Corresponding author. of fda in the genome, based on hybridization of Aldol.
7495
7496 NOTES J. BACTERIOL.

EcoRV
1 AAQATATUATTTTGTTGAATAGGTCCTTCCTTTAATACACGAAAATACCATCCAGTTCTTCCAGTTTCTTGGATACGTCTTCCCCATTCAATCCAACGCA

101 CACGTCTCCCTGGCTTCCAACAAGGTCTGCGCGGTTGTGAAACTTGTACAGTCGCTCCTCCGATTTGATAGATGTCACCAATATAAACAGAAGTTTCATC

201 CATATCAATTACGGAAACATTCTCTCCATTCAACCCTGGTTTAAAATGAACITCCGGATATTCTACTGTCCATTTTTCATAATGCGATTTACTATAAGCA

301 AATAATGCTTTTTCTGGTCCGCCATGATGTTTTTTCTCCCCGACATCATCACCAACTAAACCTGTTTTCGTTAGTAAAACCGCTTGGTCTGTCGGATTTT
SalI
401 TAAACGCTGCTGTCGACCATTCTTGTTCTAAAGGATTTTCAGCATTCGGATCACCTACTTTTTGAATGCCGCCTCTAAATAATTGCTCGATTTGACTCAC
-35
501 TTGTTCCACCCTCTCTCAAAGCTATTATAACGACTTTTTGACAAATTCTTAAGAGTAAGCGTTTTTTTTAATTTTTTTAAAGTAAAGTA¶AATC
+1
-10 I S.D.
601 AATTATTTTTTTCCATACAGACATTGAAAGAGAAAAGCAAAATGCGGAAAGArTCAATAAATGAACCAAGAACAATTTGACAAAATTAAAAATG
M N Q B Q F D K I K N 11

701 GTAAAGGATTTATCGCAGCATTGGACCAAAGCGGTGGTAGTACTCCGAAAGCGCTAAAAGATTATGGCGTTGAAGAAAATGAATACTCTAACGATGAAGA
G K G F I A A L D Q S G G S T P K A L K D Y G V E E N E Y S N D E E 45

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801 AATGTTCAACCTTGTACACGATATGCGTACTCGTATCATTACTTCACCTGCATTTAACGGAGAAAAAATCTTAGGTGCGATTCTATTCGAACAAACTATG
M F N L V H D M R T R I I T S P A F N G E K I L G A I L F E Q T M 78
SalI
901 GACCGTGAAGTTGAGGGCAAATACACAGGTTCATATTTAGCAGATAAAGGTATCGTTCCATTCTTGAAAGTCGACAAAGGTTTGGCTGAAGAAGCTGACG
D R E V E G K Y T G S Y L A D K G I V P F L K V D K G L A E E A D 111

1001 GCGTTCAATTAATGAAACCTATTCCAGACTTAGATAAATTATTAGATCGTGCGAACGAACGTGGTATCTTCGGTACTAAAATGCGTTCTAACATCTTAGA
G V Q L M K P I P D L D K L L D R A N E R G I F G T K M R S N I L E 145
PvuII
1101 AAATAATAAAGAAGCAATTGAAAAAGTTGTTAAACAACAATTTGAAGTTGCAAAAGAAATCATTGCAGCGTCTAGTACCAATTATCGAACCTGAAGTT
N N K E A I E K V V K Q Q F E V A K E I I A A G L V P I I E P E V 178

1201 AACATCAATGCTAAAGACAAAGAAGCTATCGAAGCTAACTTAGCTGAAGCAATCAAAGCTGAATTAGATAACTTGAAAAAAGATCAATATGTAATGTTGA
N I N A K D K E A I E A N L A E A I K A E L D N L K K D Q Y V M L 211

1301 AATTAACTATTCCAACTAAAGTGAATGCTTACAGCGAATTAATTGAACATCCACAAGTAATCCGCGTGGTTGCATTATCTGGTGGTTACAGCCGCGACGA
K L T I P T K V N A Y S E L I E H P Q V I R V V A L S G G Y S R D E 245
HindIII EcoRI
1401 AGCAAACAAAATCTTGAAACAAAATGATGGTTTAATCGCA=G CTCACGTGCATTAGTATCTGACTTAAACGCACAACAATCAGATGCA AT
A N K I L K Q N D G L I A S F S R A L V S D L N A Q Q S D A E F N 278
Clal Hindlll HindlIl
1501 GAAAAATTACAAGAAGCTATCGATACAATCTTCGATGCTTCAGTAAACAAAGCTAATCTAAGCTTAAAATAATAACGAGCCCC TTC TTGAGGGCT
E K L Q E A I D T I F D A S V N K A 296

1601 CGCTTTTTTGTATAATTCCATAGATTTTCTCCGCAAATTTTCTCATACGTAATATTTTCACAATAAAAGTTCTTTTCTTTCACTGTCATACATAATAAAA

1701 TACAAGAGACTTTCAGAAATAAAGAACTAAGTCGCCAAACTTACAACAGGATCTGAACACACATGGCGTCTAATCCTATTCCTCGTTATTTACAAGATAA

1801 GCGCGCAAATTTCCATGAACTATTTTATATTAATATTTGTATATGCCTTACAAAAAATTGCGCATGTGCTTTTACATACTGAAAAAATGGCACACTTT
Xba I
1901 CCTAGA

FIG. 2. Nucleotide sequence of thefda-containing 1.9-kb EcoRV-XbaI fragment of S. carnosus TM300. The transcriptional start is marked with
+1. The -35 and -10 regions and the potential ribosome binding sequences (S.D.) are underlined. The determined N-terminal protein sequence
is marked with boldface letters. The Schiff base-forming lysine residue is marked with an asterisk. The terminator is underlined with arrows.

NQEQFDK (18a), we synthesized (Gene Assembler Plus; Chromosomal DNA from S. carnosus (29) was prepared by a
Pharmacia LKB) the wobble oligonucleotide Aldol: modification of the cleared-lysate method (22). Colonies
GAT AA
TTT grown on one B plate (10 g of casein hydrolysate 140 [GIBCO,
AAT CAA
GAA CA-A
AAT CAA
C G
CAA
C GC GAAG Eggenstein-Leopoldshafen, Germany],
[Difco], 5 g of NaCl, 1 g of glucose, 1 g 5of gK2HPO4, extract
of yeastand 12 g
The oligonucleotide was labeled with [_y-32P]ATP (Amersham) of agar [GIBCO] per liter [pH 7.3]) were resuspended in 1 ml
with T4 polynucleotide kinase (Stratagene) (27). Unincorpo- of NT buffer (100 mM NaCl and 20 mM Tris-HCl, pH 7.5).
rated nucleotides were separated from the radiolabeled oligo- After incubation with 14 U of lysostaphin (Sigma, Deisen-
nucleotide by using the Nuc Trap Push Columns (Stratagene). hofen, Germany) for 30 min at room temperature, the cells
VOL. 175, 1993 NOTES 7497

human A: 215 - K A L S r H H I Y L E G T L L P N M V A G-237 A.


human B: 215- K A L N IDH H V Y L E G T L L K P N M V A G-237 2.5 10
humanC: 214- K A L N DIH H V Y L Q G T L L K P D M V P G-236
dros.mel.: 212- K A L S H H V Y L Q G T L L P N M V A G-234
2
zea mays: 2 09 - KAL N E H H V L L E G T L L R P N M V P G-232
E
S. aureus: KGL A S E Q D D V -vML K P I N L D E
0
E 1.5
S.carnosus: 197- AE LEN L K K D YV ML K L T I P K V-219

FIG. 3. Alignment of class I aldolase sequences around the pro-


go
00
-J

posed active site. The amino acid sequence of S. carnosus FBP


aldolase, deduced from thefda nucleotide sequence, is shown aligned 0.1
with the corresponding human aldolase A (17), human aldolase B (26),
human aldolase C (24), D. melanogaster (dros.mel.) (3), Z. mays (18), 0.5
and S. aureus (9) sequences. DNA and protein sequences were
analyzed by using the computer programs of Microgenie (Beckman)
and PC/Gene (IntelliGenetics, Inc.). Amino acids identical to those in 0 0.01
the S. carnosus sequence are boxed. 0 1 2 3 4 5 6 7 8

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t (h)
were lysed by the addition of 110 ,ul of cold lysis buffer (50 mM
Tris-HCl, 300 mM EDTA, 0.5% Brij 58, 0.04% Na-desoxy-
cholate, pH 8.0) on ice for about 1 h. The proteins were
removed by proteinase K digestion. DNA was separated by
B.
CsCl centrifugation.
Established protocols (27) were followed for molecular
biological techniques unless otherwise stated. Isolated chromo-
somal DNA was digested with several restriction enzymes. 4h 5h 6h 7h 8h
DNA fragments were blotted (32) onto nylon membranes A B A B A B A B A B S
(Pall; BiodyneA, 0.2-,um pore size, unloaded) by using a kd
vacuum blotting system (Pharmacia LKB) according to the - 97.4
instructions of the manufacturer. DNA was cross-linked with a - 66.2
UV-Stratalinker (Stratagene). Prehybridization was performed - 45.0
at 42°C for at least 2 h with 50 jig of heparin (Sigma) per ml as
a blocking agent. Hybridization was performed with 10 pmol of -
31.0
labeled Aldol at 42°C for at least 12 h to identify fda-
containing fragments. To remove unbound oligonucleotides,
filters were washed for 30 min at room temperature and for 10 - 21.5
min at 50°C. Nylon membranes were exposed to an RX NIF
18 x 24 film (Fuji) at - 70°C with an intensifying screen - 14.4
(Dupont).
The hybridization pattern of Aldol (Fig. 1A) allowed the
construction of a restriction map (Fig. 1B). The smallest FIG. 4. (A) Comparison of aldolase activity in S. carnosus wild type
fragment which hybridized with the probe was a 2.2-kb EcoRI- andfda clone. Black squares, S. carnosus TM300(pRB473fda20); white
PvuII fragment. squares, S. carnosus TM300; triangles, growth curve of S. carnosus
Cloning and sequencing offda from S. carnosus. Since it was TM300 and S. carnosus TM300(pRB473fda20). OD, optical density.
possible that Aldol hybridized very near the EcoRI or the (B) Coomassie blue-stained SDS-PAGE. Proteins of cell extracts of S.
carnosus TM300 (lanes A) and S. carnosus TM300(pRB473fda20)
PvuII site, we cloned the larger encompassing 5.2-kb PstI (lanes B), prepared at the indicated times, were separated on an
fragment. An enriched gene library was made by digesting SDS-12.5% polyacrylamide gel. The aldolase band is marked by an
chromosomal DNA with PstI. Fragments of 4.5 to 6.5 kb were arrow. Molecular mass standards were obtained from Bio-Rad.
isolated and ligated into PstI-digested pBluescriptll KS+
(Stratagene) and transformed into E. coli SURE (Stratagene).
One thousand transformants with inserts were analyzed by
hybridization with Aldol. Two positive clones, both having the DNA sequencing of the 1.9-kb EcoRV-XbaI fragment re-
supposed restriction map, were found. The plasmid containing vealed one open reading frame of 888 nucleotides encoding a
the 5.2-kb PstI fragment was designated pBluescriptll-fdalO. protein with an Mr of 32,855 (Fig. 2). The deduced N-terminal
The approximate position of fda was determined by exonucle- amino acid sequence corresponds exactly with that determined
ase III digestion and subcloning (Fig. 1B). by Edman degradation of the purified aldolase (3a). The fda
The insert was sequenced by double-stranded DNA se- stop codon is followed by an inverted repeat sequence (AG =
quencing (7) by using the dideoxy procedure (28), the Phar- - 29.2 kcal [ca. - 122 kJ]) (34) which is indicative of a
macia AutoRead Sequencing kit, and the A.L.F. DNA Se- rho-independent transcription terminator sequence (Fig. 2).
quencer from Pharmacia LKB. Exonuclease III clones were Transcriptional start. The transcriptional start of fda was
sequenced with universal and reverse primers obtained with determined by primer extension analysis. Total RNA from S.
the sequencing kit. Other sequencing primers were synthesized carnosus was isolated by the guanidine isothiocyanate method
by a synthesizer (Pharmacia) and labeled with fluorescein as previously described (8) with some modifications. The
amidite. culture was incubated at 37°C in B broth to an A578 of 2.2. The
7498 NOTES J. BACTERIOL.

cells were harvested and disrupted with glass beads (0.1- to phate dehydrogenase and triosephosphate isomerase as de-
0.3-mm diameter; Fluka) as previously described (31). The scribed by the manufacturer (Sigma).
supernatant was loaded onto a Cs-trifluoroacetate gradient (p S. carnosus(pRB473fda2O) had an aldolase activity six- to
= 1.51 g/ml). The RNA was pelleted by ultracentrifugation. eightfold higher than that of the wild type. This observation is
RNA (10 ,ug), 0.1 pmol of 5'-end-labeled oligonucleotide corroborated by SDS-PAGE, in which a higher protein con-
Aldo2 (5'-CCG CTrL TGG TCC AAT GCT GCG ATA AAT centration was visible at the expected size of 33 kDa with S.
CCT TTA CC-3') (corresponding to nucleotide positions 734 carnosus TM300(pRB473fda2O). The specific aldolase activity
to 699 in Fig. 2), and 10 U of avian myeloblastosis virus reverse was nearly constant during the course of exponential growth
transcriptase (Stratagene) were used for the primer extension (Fig. 4A).
experiments. Extension products of DNA sequencing reactions It was suspected that the glucose concentration in the
were resolved on a denaturing 6% polyacrylamide gel and growth medium influenced aldolase production. S. carnosus
visualized by autoradiography. The promoter region is similar and S. carnosus(pRB473fda2O) were therefore aerobically
to E. coli promoters; 4 of 6 bases of the fda -35 and -10 grown in B broth (without glucose) supplemented with 0.0, 0.1,
regions match the E. coli promoter consensus sequence. 0.4, 1, or 4% glucose. After 6 h, cell extracts were assayed for
Homology with other class I FBP aldolases. The deduced specific aldolase activity. In the presence of 0.4% glucose, the
amino acid sequence of the FBP aldolase of S. carnosus was specific aldolase activity was nearly twofold higher than in the
compared with other class I aldolase sequences, in the Micro- absence of glucose. With higher glucose concentrations the
genie and EMBL-Heidelberg data banks. With the S. aureus specific aldolase activity was decreased. Anaerobic growth
FBP aldolase the amino acid sequence around the active site conditions had no significant influence on specific aldolase

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lysine residue was determined (9). This sequence is highly activity in both the wild type and S. carnosus(pRB473fda2O).
conserved not only in the S. carnosus but also in other FBP Nucleotide sequence accession number. The sequence offda
aldolases (Fig. 3). The amino acid identity was 33.5% with has been submitted to the GenBank data base and assigned
human aldolase B (covering 184 amino acids [aa]), 33.7% with accession number X71729.
human aldolase C (covering 175 aa), 25.1% with Drosophila
melanogaster (covering 215 aa), 25% with Zea mays (covering We thank M. R. Kula and H. P. Brockamp (Institut fur Enzymtech-
155 aa), 27.5% with Oryza sativa (covering 153 aa), and 36.9% nologie der Universitat Dusseldorf, Forschungszentrum Julich) for the
with Plasmodium falciparum (covering 65 aa). S. carnosus FBP N-terminal aldolase sequence information, Karen A. Brune for criti-
aldolase showed no homology with the class II FBP aldolase cally reading the manuscript, and Arielle Ferrandon, Vera Augs-
(1) or the 2-keto-4-hydroxyglutarate aldolase (23) of E. coli. burger, and Elisabeth Knorpp for expert technical assistance.
This work was supported by grants from the Deutsche Forschungs-
Complementation of E. coli NP315 (Fda-). The mutant E. gemeinschaft (SFB 323).
coli NP315 (Fda-), a derivative of strain K-10 with a temper-
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