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J Bacteriol, April 1998, p. 2212-2219, Vol. 180, No. 8
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Molecular Analysis of the Gene Encoding
F420-Dependent Glucose-6-Phosphate Dehydrogenase from
Mycobacterium smegmatis
Endang
Purwantini
and
Lacy
Daniels*
Department of Microbiology, University of
Iowa, Iowa City, Iowa 52242
Received 11 September 1997/Accepted 6 February 1998
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ABSTRACT |
The gene fgd, which codes for
F420-dependent glucose-6-phosphate dehydrogenase (FGD), was
cloned from Mycobacterium smegmatis, and its sequence was
determined and analyzed. A homolog of FGD which has a very high
similarity to the M. smegmatis FGD-derived amino acid
sequence was identified in Mycobacterium tuberculosis. FGD
showed significant homology with F420-dependent
N5,N10-methylene-tetrahydromethanopterin
reductase (MER) from methanogenic archaea and with several
hypothetical proteins from M. tuberculosis and
Archaeoglobus fulgidus, but FGD showed no
significant homology with NADP-dependent glucose-6-phosphate
dehydrogenases. Multiple alignment of FGD and MER proteins revealed
four conserved consensus sequences. Multiple alignment of FGD with the
hypothetical proteins also revealed portions of the same conserved
sequences. Moderately high levels of FGD were expressed in
Escherichia coli BL21(DE3) carrying fgd in
pBluescript.
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INTRODUCTION |
Each year, there are ~10 million
new cases of tuberculosis in the world (mostly in developing
countries), one-third of which cause death (49). In the
developed world, ~50% of AIDS patients ultimately suffer from
debilitating Mycobacterium avium infections (4,
26). However, there are major problems in the
treatment of these diseases due to inherent or acquired drug
resistance and to serious side effects of the drugs used (4, 5, 8, 22, 26). We have begun to examine coenzyme
F420-dependent reactions in mycobacteria to
determine if such reactions are important for this group of
pathogens. If so, specific enzymatic steps can be identified as targets
for chemotherapy. It is likely that drugs aimed at
F420-related targets would act by mechanisms completely different from those of the more widely used drugs.
Coenzyme F420 is a two-electron transfer coenzyme. It was
first discovered in methanogenic archaea (14, 15), where it is involved in several reactions in methane biosynthesis. At least five
methanogenic enzymes are F420 dependent:
F420-dependent methylene-tetrahydromethanopterin dehydrogenase (24, 39, 40),
methylene-tetrahydromethanopterin reductase (MER) (34, 60),
formate dehydrogenase (29, 53), F420-reducing
hydrogenase (19, 28), and alcohol dehydrogenase (6,
61). In nonmethanogenic archaea, F420 is found in
Halobacterium (13, 33), Thermoplasma
(33), Sulfolobus (33), and
Archaeoglobus (38) species. In the bacterial
domain, F420 has been found in Streptomyces
species (10, 12, 37), Anacystis nidulans
(18), Nocardia aurantia (12), and
several Mycobacterium species (12, 41, 47).
Scenedesmus acutus, a green alga and a member of the domain
Eucarya, also contains F420 (17).
Some steps in tetracycline (37) and lincomycin
(10) biosynthesis by Streptomyces
species require F420. Coenzyme F420 is a
component of the DNA repair photolyase in several microorganisms
(16, 17, 30, 36).
Although the presence in Mycobacterium species of an unknown
compound with spectral properties very similar to those of
F420 was reported in 1960 (11), and
F420 was clearly identified in these organisms in the 1980s
(12, 41), any role of this coenzyme in these organisms
remained unknown until recently. A study to determine the function
of F420 in Mycobacterium smegmatis led us to the
discovery of a novel glucose-6-phosphate dehydrogenase which
specifically uses F420 as its electron acceptor
(47). This enzyme was named FGD, for
F420-dependent glucose-6-phosphate dehydrogenase. Not all
F420-containing organisms possess FGD. So far, FGD has
been found only in Mycobacterium and Nocardia species and in Gordona amarae (48).
We also observed that in addition to FGD, Mycobacterium
species possess an NADP-dependent glucose-6-phosphate dehydrogenase (47, 48), which is consistent with previous reports (2, 3). NAD- or NADP-dependent glucose-6-phosphate dehydrogenases are
commonly found in many organisms and are named ZWFs (for
zwischenferment) (20). Many zwf genes from
bacteria, yeasts, animals, and humans have been cloned and sequenced
(21, 32, 42, 44, 51). The deduced amino acid sequences of
these enzymes have been compared, and conserved regions have been
identified. There is high homology between ZWF amino acid sequences
from diverse sources. Conserved regions have been hypothesized to be
sites for coenzyme (NAD or NADP) and substrate (glucose-6-phosphate)
binding and for catalysis. We were interested to know the structural
relationship between ZWFs and FGD since these two types of enzymes
catalyze similar reactions, differing principally in the electron
acceptor used. By using the NH2-terminal amino acid
sequence of the purified FGD, a mixture of oligonucleotides was
designed and used as a probe to isolate the gene for FGD. Here we
describe the molecular characterization of fgd from M. smegmatis mc2155 and its functional expression in
Escherichia coli.
(A preliminary version of some of this work has been presented in a
poster format [46].)
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MATERIALS AND METHODS |
Chemicals and enzymes.
Restriction enzymes, T4 DNA ligase,
and calf intestine alkaline phosphatase were purchased from New England
Biolabs (Beverly, Mass.). The oligonucleotide labeling kit Genius no.
3, alkaline phosphatase-conjugated antidigoxigenin antibody, nitroblue
tetrazolium (NBT), 5-bromo-4-chloro-3-indolylphosphate (BCIP), and
DNase-free RNase were from Boehringer Mannheim Biochemicals
(Indianapolis, Ind.). DNA molecular weight markers (1-kb ladder)
and TRIzol were from Gibco-BRL Life Technologies (Grand Island, N.Y.).
Qiagen tips (for plasmid preparation) and Qiaquick columns and
accessories (for purification of DNA fragments from agarose gels) were
from Qiagen Inc. (Chatsworth, Calif.). Coenzyme F420 was
purified from Methanobacterium thermoautotrophicum Marburg,
using 70% ethanol extraction at 4°C and DEAE and C18
column chromatography (45).
Bacterial strains, plasmids, media, and growth conditions.
M. smegmatis mc2155 (a gift from W. Jacobs, Jr.,
Albert Einstein College of Medicine, New York, N.Y.) was grown in
Middlebrook 7H9 medium supplemented with 0.2% glycerol and 0.05%
Tween 80. For cloning and sequencing, E. coli XL1-Blue and
pBluescript II SK+ (Stratagene, La Jolla, Calif.) were used as
recombinant host and vector, respectively. E. coli BL21(DE3)
was used as a host for protein expression. E. coli strains
were grown at 37°C on solid medium or in liquid Luria-Bertani (LB)
medium. Ampicillin (100 µg/ml) was incorporated into LB medium to
select for recombinants. For protein expression studies, E. coli strains were grown as described before (57), with
0.4 mM isopropyl-
-D-thiogalactopyranoside (IPTG) as an
inducer.
Molecular biology techniques.
M. smegmatis chromosomal
DNA was purified as described by Husson et al. (27).
Recombinant plasmids from E. coli XL1-Blue were purified by
using Qiagen tips as instructed by the manufacturer. Restriction
enzymes and DNA-modifying enzymes were used according to the
manufacturer's protocols. Standard molecular biology protocols for
cloning, subcloning, and protein expression in E. coli
(52) were used throughout.
Southern and colony hybridization was performed as specified by the
manufacturer (Boehringer), using a completely degenerate oligonucleotide, 5'GCRAAYTGYTCNGCNGANGCYTTRTANCCNAGYTT3'
(R = A + G, Y = C + T, N = A + G + C + T), complementary to the NH2-terminal amino acid
sequence from residues 3 to 15 of FGD (LKLGYKASAEQFA). This 35-mer was
labeled at its 3' end by digoxigenin-ddUTP, using a Genius labeling
kit. Prehybridization and hybridization were at 50°C, and
posthybridization washes were at room temperature. Hybridizing bands or
colonies were detected by alkaline phosphatase-conjugated antidigoxigenin antibody and colorimetric substrates NBT and BCIP.
A combination of subcloning and primer walking methods was used for DNA
sequencing. Universal primer (SK+) was used for the
first round of
sequencing. DNA sequencing was done at the University
of Iowa DNA
Facility by using an Applied Biosystems model 373A
DNA sequencer.
Insertional mutagenesis of cloned
fgd was conducted by using
a kanamycin resistance (Kan
r) cassette (Pharmacia Biotech,
Piscataway, N.J.).
Computer analysis of sequences.
Sequence comparisons of the
derived amino acid sequences to entries in the protein database were
performed by using the BLASTP program of the National Center for
Biotechnology Information (NCBI) (1). When appropriate,
sequences were compared with each other by using the BestFit and PileUp
programs of the Genetics Computer Group package and the ClustalW and
Pima programs at the Human Genome Center at the Baylor College of
Medicine (54, 58).
Detection of FGD activity in recombinant E. coli.
Cell
extracts of E. coli strains were prepared by cell breakage
in a French pressure cell (5,000 lb/in2). The resulting
cell lysates were centrifuged at 10,000 × g for 15 min. The proteins from these extracts were precipitated with ammonium
sulfate at 90% saturation (0°C) and resolubilized in 20 mM Tris HCl
(pH 7.0). These solutions were assayed to determine FGD activities and
protein content as described before (47).
Nucleotide sequence accession number.
The nucleotide
sequence data reported here appear in the GenBank nucleotide sequence
database under accession no. AF041061.
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RESULTS AND DISCUSSION |
Cloning, subcloning, and sequencing of M. smegmatis
fgd.
Southern analysis of M. smegmatis DNA that
was completely digested with BamHI, using the
degenerate oligonucleotide complementary to the
NH2-terminal sequence of FGD, showed a positive
hybridization signal of ca. 7 kb. Accordingly, M. smegmatis
DNA was digested completely with BamHI and electrophoresed
in a 0.8% agarose gel; DNA fragments of ca. 6 to 8 kb were recovered
and purified by using Qiaquick columns. These DNA fragments were
ligated into BamHI-digested, dephosphorylated pBluescript II
SK+. The ligation mixture was used to transform competent cells of
E. coli XL1-Blue, and recombinants were screened for an
fgd clone by colony hybridization. Two colonies out of ca.
400 screened showed positive hybridization signals, both of which
carried plasmids with inserts of the same size and restriction
patterns. One of these two recombinant strains was preserved and used
for further work, and the corresponding plasmid was designated pEP7000.
The restriction map of the 7-kb fragment obtained by using various
restriction enzymes is shown in Fig.
1A. Shown in Fig. 1B are the maps of
various subclones described in more detail below.
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FIG. 1.
Restriction maps of pEP7000 and various subclones.
Symbols: B, BamHI; Bg, BglII; P, PstI;
A, AccI; S, SmaI; aph, aminoglycoside
phosphotransferase, which imparts Kanr; fgd,
F420-dependent glucose-6-phosphate dehydrogenase gene;
orfR, possible regulatory gene. Restriction sites at the
ends of the subclone fragments indicate the boundary of M. smegmatis DNA in the plasmid inserts, not necessarily the
restriction sites used for the final subcloning step (see text for
descriptions of construction).
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The 1.1-kb
SmaI fragment was subcloned into the
SmaI site of pBluescript, giving plasmid pEP1088. This
plasmid was used for
the first round of sequencing. Further sequencing
was performed
by primer walking using pEP7000. The gene for FGD was
located
ca. 2 kb away from one end of the 7-kb insert of the primary
clone
and in the same direction as and downstream of the T7 promoter
of
the vector.
In addition to pEP1088, three more subclones were developed by using
pBluescript II SK+ (Fig.
1B): pEP1300, carrying the 1.3-kb
AccI/
PstI fragment (
fgd with 244 bp
upstream and 36 bp downstream);
pEP3000, carrying the 3-kb
PstI fragment of pEP7000 (
fgd with
ca. 2,000 bp
upstream and 36 bp downstream; the downstream
PstI
site is
shown in Fig.
1B, and the upstream
PstI site is in the
pBluescript multicloning site); and pEP5000, containing the equivalent
of the 5-kb
SmaI/
BamHI fragment of pEP7000, which
was constructed
by the ligation of the 0.8-kb product of an
EcoRI/
BglII double
digest of pEP1088 and the
7.3-kb fragment of an
EcoRI/
BglII double
digest
of pEP7000 (
fgd with 250 bp upstream and approximately
3.9 kb downstream; the
EcoRI site is upstream of
fgd
in the multicloning
site, and the
BglII site is in
fgd). These plasmids were used
for enzyme expression
studies.
Southern hybridization of
M. smegmatis DNA, which had been
cut with various restriction enzymes, with oligonucleotide probe
that
precisely complemented the 5' end of
fgd showed one
hybridizing
band from each restriction digest, which indicates that
only one
copy of
fgd was present in the
M. smegmatis genome. The sequence
of
fgd from
M. smegmatis and its flanking regions are shown in
Fig.
2. The gene
fgd consisted of
1,008 bases, corresponding to
336 amino acid residues. The
NH
2-terminal amino acid sequence
of purified FGD
(
47) precisely matched the deduced amino acid
sequence of
the NH
2 terminus, with alanine as the first amino
acid.
This finding indicates that the initiation codon was GTG,
which codes
for valine. Thus, the ATG which immediately precedes
the GTG is not
translated. TGA was the stop codon. In
E. coli,
GTG rarely
functions as an initiation codon, and when it does
it is less efficient
than ATG (
23). However,
Mycobacterium species
use
GTG as the initiation codon for many genes (
9,
25,
35,
59).
The G+C content of
fgd was 65%, which is in the range of
the G+C content of
Mycobacterium chromosomes (62 to 70%).
The
FGD molecular mass predicted from the deduced amino acid sequence
was 37,148 Da, slightly lower than the subunit molecular mass
of
ca. 40,000 Da determined for the purified
M. smegmatis
protein
by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE)
(
47). The theoretical pI value
for FGD was 5.20.
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FIG. 2.
Nucleotide sequence of fgd and some of its
flanking regions, and deduced amino acid sequences of FGD and the
possible regulatory protein ORFR. Underlined,
NH2-terminal amino acid sequence determined from purified
FGD;  , direction of fgd transcription;  , direction of
transcription of genes adjacent to fgd.
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Comparative sequence analysis.
The complete amino acid
sequence of FGD showed a very high homology (BLAST score,
1,592; probability, 4.4e
211) with a hypothetical protein
(g1817673) from Mycobacterium tuberculosis (1). BestFit analysis of these two proteins showed 89%
identity and 93% similarity, providing strong evidence that this is
FGD in M. tuberculosis, given that FGD activity is present
in M. tuberculosis (48). A BestFit analysis of
the two fgd nucleotide sequences showed 80% identity.
While writing this report, we became aware of related work. A gene that
was involved in the activation of an experimental
antituberculosis drug
called NAP (nitroimidazole pyran; also called
PA-824) in
Mycobacterium bovis BCG had been discovered and sequenced
(
55,
56). If this gene is deleted or mutated,
M. bovis BCG
becomes NAP resistant. The derived amino acid sequence
of the
product of the gene that complemented NAP resistance of
M. bovis BCG was identical with that of FGD from
M. tuberculosis (
56).
Thus, FGD is involved in some way in
the activation of NAP.
No good sequence homology was observed when the complete amino acid
sequences of ZWFs from
E. coli,
Leuconostoc
mesenteroides,
and human cells were individually compared with
that of FGD by
using the BestFit program. The percent identities of FGD
with
these ZWFs were 17 to 19%, much lower than the average percent
identities seen between ZWFs themselves. For example, Rowland
et al.
showed that a multiple alignment of 13 known ZWF sequences
from various
organisms has a mean conservation of 39% (
50).
A multiple
alignment of FGD from
M. smegmatis with ZWF gene products
from
E. coli,
L. mesenteroides,
Saccharomyces cerevisiae, and
human cells showed very little
sequence conservation, and in particular
key conserved regions found in
all ZWFs were not present in the
few areas of FGD that do align with
ZWF (data not shown).
When the FGD sequence was compared by BLAST analysis with protein
sequences in the NCBI databank, regions of this protein
showed homology
with the F
420-dependent MER from several methanogens
(
7,
43,
60) and
Archaeoglobus fulgidus
(
31). FGD also
had regions that showed homology to at least
eight hypothetical
proteins from
M. tuberculosis and one
from
A. fulgidus and to
the product of the
lmbY
gene of unknown function from
Streptomyces lincolnensis.
Aside from the
M. tuberculosis and
M. bovis FGD
homologs, an unknown hypothetical protein from
M. tuberculosis (1877257) showed the highest similarity, with an
identity of 37%.
The percentage identities of FGD with MERs were 23 to
28%. The
other group of
M. tuberculosis hypothetical
proteins had identities
of 24 to 29%. These values were all higher
than the percent identities
seen between FGD and several ZWFs (17 to
19%). Similarities with
other F
420-utilizing enzymes were
not seen in the BLAST analysis,
except for the short
NH
2-terminal fragment of the
F
420-dependent
alcohol dehydrogenase from
Methanogenium liminatans (
6); unfortunately,
the
full sequence of this protein is not yet known.
Both ClustalW and Pima multiple alignments of FGD were conducted with
MER proteins, most of the homologous unknown proteins
identified by the
BLAST search, and four F
420-using enzymes
(methylene-tetrahydromethanopterin
dehydrogenase, hydrogenase
subunit, formate dehydrogenase
subunit, and photolyase). FGD and
MER showed significant homology
in alignments using both programs,
characterized by four conserved
segments (identified by ClustalW) that
are shown in Fig.
3. These
segments were
in the NH
2-terminal two-thirds of the molecule.
FGD-MER
consensus sequence 2 identified MER and several of the
mentioned
unknown proteins in a BLAST search of the NCBI database.
Sequence 4 identified MER and one unknown protein, while sequence
3 identified
only MER. Sequence 1 did not identify homologous
sequences, probably
due to its short length. Consensus sequences
1 to 4 did not identify in
a BLAST analysis any known enzymes
that do not use F
420 as
a coenzyme. Areas with conserved amino
acids in the carboxy one-third
were less similar than the four
identified in the
NH
2-terminal region and are not underlined in
Fig.
3; they
may be of significance, but their consensus sequences
did not lead to
the identification of any proteins in BLAST searches.
Aside from MER
and the alcohol dehydrogenase, no F
420-utilizing
enzymes
that had similarities to FGD were detectable by ClustalW
or Pima
multiple alignments.
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FIG. 3.
Multiple alignments of FGD with MER. Identical amino
acids are highlighted in black, and similar amino acids are highlighted
in grey. The solid bar below the consensus sequence indicates areas of
significant similarity, corresponding to FGD-MER consensus sequences 1 through 4 as labeled adjacent to each bar. For presentation purposes,
the eight NH2-terminal amino acids of Methanococcus
jannaschii MER were not included in the alignment. ClustalW
(58) was used to align the sequences, and Boxshade (0.9 setting) was used to determine the degree of residue shading.
Sequences, from top to bottom: Methanococcus jannaschii MER
(GenBank accession no. E64491), Methanobacterium
thermoautotrophicum MER (S66529), A. fulgidus MER
(2649522), Methanopyrus kandleri MER (1002714), M. tuberculosis FGD (e301455), and M. smegmatis FGD
(AF041061).
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When FGD was compared by multiple alignment to seven unknown proteins
(six from
M. tuberculosis and one from
A. fulgidus)
that had been identified in a BLAST search, consensus
sequences
similar to those of the FGD-MER alignments were found. Figure
4 presents an abbreviated alignment with
the most relevant and
most homologous sections shown. FGD-unknown
consensus sequence
2 was the strongest in similarity to an FGD-MER
consensus sequence
and led to the identification of FGD, MER, and
unknown proteins
in a BLAST search. The other consensus sequences in
this comparison
were not as strong in their similarity to the FGD-MER
comparison.
We hypothesize that a conserved site important in
F
420-dependent
catalysis is defined by one or more of the
four FGD-MER consensus
sequences in Fig.
3 and that other
F
420-dependent enzymes not
similar to FGD (hydrogenase,
formate dehydrogenase, methylene-tetrahydromethanopterin
dehydrogenase,
and photolyase) have a different type of F
420-reactive
site. We also hypothesize that the unknown hypothetical proteins
in
M. tuberculosis interact with F
420; the
number of these proteins
(six) suggests that study of F
420
metabolism in mycobacteria may
be very fruitful in the discovery of
novel enzymes. Proof of our
hypotheses concerning conserved sites for
interaction with F
420 will require considerable further
biochemical examination of MER,
FGD, and the unknown proteins.
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FIG. 4.
Multiple alignments of FGD and unknown hypothetical
proteins initially identified by BLAST search with FGD. Identical amino
acids are highlighted in black, and similar amino acids are highlighted
in grey. The solid bar below the consensus sequence indicates areas of
significant similarity which correspond to FGD-MER consensus sequences
1 through 4 shown in Fig. 3. For presentation purposes, short sections
of NH2- or carboxy-terminal amino acid sequences of
proteins are not included in the alignments. ClustalW (58)
was used to align the sequences, and Boxshade (0.8 setting) was used to
determine the degree of residue shading. Sequences, from top to bottom:
M. tuberculosis FGD (e301455), M. smegmatis FGD
(AF041061), M. tuberculosis unknown proteins (1877257, 1403414, 1781192, 1694883, 1524222, and 1723065), and A. fulgidus unknown protein (2650688).
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Identity of sequences adjacent to fgd.
Analysis of the
sequence downstream of fgd showed an open reading frame
(called orfR) encoding a hypothetical protein (ORFR) of 254 amino acids which should be transcribed in the direction opposite that
of fgd (Fig. 1). The DNA and corresponding protein sequences
of ORFR are shown in Fig. 2. ORFR had good homology with several
hypothetical regulatory proteins of the GntR family with
helix-turn-helix motifs. The highest BLAST score (157, probability of
5.6e18) was seen with a hypothetical regulatory protein
in E. coli (accession no. P31453); there were 18 other
hypothetical regulatory proteins with BLAST scores >100. Sequence
comparison of ORFR to this protein with the BestFit program revealed an
identity of 25% and similarity of 39%. The consensus sequence of the
GntR family matched perfectly with the sequence of ORFR at positions 35 to 56 (EREMAETFAVSRSTLRSALLPL). The fact that BLAST
analysis did not reveal a highly similar protein in M. tuberculosis (for which the complete genome is available) suggests
this protein is not ubiquitous within the mycobacteria.
The sequence upstream of
fgd showed an ORF encoding a
protein of 248 amino acids which should be transcribed in the direction
opposite of
fgd. This protein showed high homology (BLAST
score
of 434, probability of 1.8e
101) to an unknown
protein from
M. tuberculosis (g1817672). The gene
for
this hypothetical protein, g1817672, was also located directly
upstream of
fgd (g1817673) in
M. tuberculosis.
The second-highest
sequence similarity (BLAST score of 134, probability of 3.3e
10) was with a
-lactamase L1
precursor from
Xanthomonas maltophilia.
Most other proteins
showing weak sequence similarities were hypothetical.
Thus, it is not
clear what this gene might code for in mycobacteria.
Expression of FGD in recombinant E. coli.
Initially,
E. coli XL1-Blue strains containing recombinant plasmids
pEP7000, pEP1300, and pEP1088 were assayed for FGD activity. The
strains carrying pEP7000 and pEP1300 expressed very low FGD activities (0.001 µmol/min/mg of protein), and no detectable
activity was observed with pEP1088. This low level of expression of
pEP7000 and pEP1300 was thought to be a result of inefficient
transcription of fgd in E. coli. To explore this
possibility, pEP7000 and pEP1300 were transformed into E. coli BL21(DE3), a lysogen that contains a T7 RNA polymerase gene
in the chromosome. As shown in Table 1,
fgd was expressed at a ca. 15- to 523-fold-higher level in E. coli BL21(DE3) than in E. coli XL1-Blue,
consistent with improved transcription in E. coli BL21(DE3).
Expression was higher with pEP1300 than with pEP7000. Induction with
IPTG increased expression only slightly. The specific
activity of
FGD in
E. coli BL21(DE3) carrying pEP1300 in the
absence
of IPTG increased as the culture aged and was highest at the
stationary
phase. We do not understand why IPTG did not result in a
higher
level expression, but this may have been due to inefficient
translation
of
fgd in
E. coli BL21(DE3). In these
constructs, translation
was expected to initiate from the mycobacterial
sequence; however,
the sequence upstream of
fgd did not show
strong homology with
typical Shine-Dalgarno consensus sequences. It has
been reported
before that there is always a basal level of T7 RNA
polymerase
in
E. coli BL21(DE3) without IPTG induction
(
57). This basal
level of T7 RNA polymerase might be enough
to express
fgd at the
levels which were observed in
noninduced cells. Thus, the translation
rate was possibly limiting for
FGD expression in
E. coli BL21(DE3).
This argument might
also explain why the increase in
fgd message
by IPTG
induction did not result in increased FGD levels. It is
also possible
that FGD expression is tightly regulated in
E. coli.
In pEP1088, a construct used for initial sequencing,
fgd was
truncated at its 3' end (deletion of 82 bp, corresponding to
deletion
of 28 amino acids at the FGD carboxy terminus). FGD was
expressed from
this construct in BL21(DE3), although the level
was only 3% of that
from pEP1300. This result indicates that this
carboxy-terminal portion
of FGD is not essential for catalysis.
Despite high FGD activities in BL21(DE3) cells, when extracts of
recombinant
E. coli BL21(DE3) were examined by SDS-PAGE,
no
intense band for FGD at ~40,000 Da was seen, indicating that
FGD was
not overexpressed. The formation of inclusion bodies by
overexpression
of FGD was not thought to be responsible for the
lack of
overexpression, since (i)
E. coli BL21(DE3) carrying pEP1300
grown at 25°C (with and without IPTG) had FGD activities that
were
twofold lower than those in the corresponding 37°C cultures,
(ii)
microscopic (phase-contrast) examination did not show any
sign of
inclusion bodies in cells which were grown in the presence
of IPTG, and
(iii) SDS-PAGE of SDS-lysed whole cells (which would
include proteins
in inclusion bodies) did not reveal bands indicative
of overexpression.
To clarify the differences in the levels of FGD expression from pEP7000
and pEP1300, we created two more plasmids (pEP3000
and pEP5000 [Fig.
1]). As shown in Table
1, the expression of
fgd from
pEP3000 was approximately the same as with pEP1300. Similarly,
pEP5000
and pEP7000 gave comparable results: ca. 15- to 25-fold
lower
activities than pEP3000 and pEP1300. Since the level of
expression from
pEP5000 was comparable to that from pEP7000, and
that from pEP3000 was
comparable to the level of expression from
pEP1300, it is possible that
the sequences downstream of
fgd,
or the corresponding gene
products, were responsible for the reduced
expression of FGD in
E. coli. This regulation might be due to
a putative
repressor protein encoded by
orfR that is immediately
downstream of
fgd; ORFR has high sequence homology with
regulatory
proteins, as discussed above. We have no data on the ability
of
this gene to affect transcription in
M. smegmatis. It is
possible
that in
E. coli, ORFR binds to a putative
mycobacterial promoter
region upstream of
fgd or to the T7
promoter region further upstream.
Further experimental evidence is
needed to determine the true
reason for the large differences in
expression seen with the different
constructs.
The cloned FGD was partially purified from
E. coli by using
ammonium sulfate precipitation (the active fraction was recovered
in
the supernatant after treatment with ammonium sulfate at 60%
of
saturation and then precipitated by ammonium sulfate at 90%
saturation) followed by desalting with a Centriplus-10 filter
and
finally F
420-affinity column chromatography
(
47). The protein
at this stage of purity had the same
denatured molecular mass,
and temperature and pH optima for FGD
activities, as the native
enzyme from
M. smegmatis. Thus,
the enzyme can be expressed in
a fully functional form in
E. coli and does not need
Mycobacterium-specific
cofactors
or other mycobacterial proteins for activity.
Insertional mutagenesis of cloned
fgd in
E. coli
was conducted by using the Kan
r gene (
aph, for
aminoglycoside phosphotransferase). The 1.3-kb
BamHI
fragment of the Kan
r cassette was inserted into the
BglII site of
fgd in pEP1300,
resulting in
pEP1300::Kan
r. No FGD activity was detected in
E. coli BL21(DE3) carrying
pEP1300::Kan
r.
| |
ACKNOWLEDGMENTS |
This work was supported by U.S. Department of Agriculture grant
4132008 to the Biotechnology Byproduct Consortium.
We are appreciative of M. smegmatis mc26,
provided by William Jacobs, Jr. We thank Biswarup Mukhopadhyay for
helpful discussions. We also thank Kendall Stover and Paul Warrener of
PathoGenesis Corporation for sharing their unpublished data.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University of Iowa, Iowa City, IA 52242. Phone: (319)
335-7780. Fax: (319) 335-9006. E-mail:
ldaniels{at}blue.weeg.uiowa.edu.
Present address: Department of Microbiology, University of
Illinois, Urbana, IL 61801.
| |
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Abstract
Introduction
