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Journal of Bacteriology, September 1998, p. 4734-4738, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Cloning, Expression, and Catabolite Repression of a
Gene Encoding 
-Galactosidase of Bacillus megaterium
ATCC 14581
Gwo-Chyuan
Shaw,*
Hsun-Sheng
Kao, and
Chih-Yung
Chiou
Institute of Biochemistry, School of Life
Science, National Yang-Ming University, Taipei, Taiwan, Republic of
China
Received 5 May 1998/Accepted 17 June 1998
 |
ABSTRACT |
A gene encoding 
-galactosidase, designated mbgA, was
isolated from Bacillus megaterium ATCC 14581. Chromosomal
-galactosidase production could be dramatically induced by lactose
but not by isopropyl--D-thiogalactopyranoside (IPTG) and
was subject to catabolite repression by glucose. Disruption of
mbgA in the B. megaterium chromosome resulted
in loss of lactose-inducible -galactosidase production. A 27-bp
inverted repeat was found to overlap the mbgA promoter
sequence. Two partially overlapping catabolite-responsive elements
(CREs) were identified within the inverted repeat. Base substitutions
within CRE-I and/or CRE-II caused partial relief from catabolite
repression. The results suggest that the 27-bp inverted repeat may
serve as a target for a catabolite repressor(s).
| |
TEXT |
Many catabolic enzymes in
Bacillus spp. are subjected to catabolite repression by
glucose or other rapidly metabolizable carbon sources (4, 11, 27,
33). The mechanism underlying catabolite repression in
Bacillus spp. is quite different from the cyclic AMP
(cAMP)-dependent positive regulatory mechanism operative in Escherichia coli (28). The latter involves a
positive regulatory protein, cAMP receptor protein (19). In
contrast, Bacillus does not contain detectable cAMP or cAMP
receptor protein-like proteins (2, 13), and addition of
exogeneous cAMP does not affect catabolite repression in B. subtilis (24). A negative transcription regulator, CcpA
(7, 9, 10), which is a member of the LacI/GalR family
(36), is believed to mediate catabolite repression by interacting with a cis-acting catabolite-responsive element
(CRE) located in the regulatory region or coding region of
catabolic-enzyme-encoding genes subjected to carbon catabolite
repression (12, 35). The consensus sequences
5'-TGWNANCGNTNWCA-3' (35), where W stands for
adenine or thymine, and 5'-(T/A)GNAA(C/G)CGN(T/A)(T/A)NCA-3' (12) have been proposed for the CRE. In this report,
we describe the cloning, sequencing, expression, and regulation of the
B. megaterium -galactosidase gene (mbgA). A
27-bp inverted repeat was identified in the mbgA promoter
region. Two CRE-like sequences within the 27-bp inverted repeat were
found to be able to exert catabolite repression on -galactosidase
production in B. megaterium.
Cloning of the -galactosidase gene.
To clone the
-galactosidase gene, chromosomal DNA of B. megaterium
ATCC 14581 was isolated as described previously (37) and was
partially digested with the restriction enzyme Sau3AI. DNA
fragments ranging in size from 6 to 9 kb were gel purified and ligated
into BamHI-cut and dephosphorylated vector pQE30 (Qiagen, Inc.). E. coli lac mutant JM109 (29) was
transformed with the ligated DNA to generate a genomic library. The
-galactosidase gene was isolated by direct selection for enzyme
activity on
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal)
plates. One plasmid isolated from a blue colony was found to contain an
insert of about 9 kb and was subjected to further analysis. A 5-kb
PstI fragment derived from the 9-kb insert was further
subcloned into PstI-cut pQE30 in both orientations. Both of
the resulting plasmids, when introduced into E. coli JM109, still gave blue colonies on Luria-Bertani (LB) medium plates
(29) containing X-Gal. These results suggest that the 5-kb
PstI fragment contains the cloned -galactosidase gene and
the promoter-like sequences from which transcription can start in
E. coli.
The 5-kb PstI fragment was further cloned into
PstI-cut pBluescript KS(+) (Stratagene, Inc.) in both
orientations for DNA sequencing by the dideoxy-chain termination method
(30). The restriction map is shown in Fig.
1. One complete open reading frame (ORF)
and two incomplete ORFs were identified. Based on its sequence homology
to several published -galactosidase genes, this complete ORF is
assumed to be the structural gene for the -galactosidase of B. megaterium and is designated here mbgA
(megaterium -galactosidase gene). This position of
mbgA in the 5-kb PstI fragment is reinforced by
assaying for changes in the -galactosidase activity of various
deletion derivatives of the PstI fragment shown in Fig. 1.
pGS240, a modified form of pBluescript KS(+) which loses
-complementation ability (29), was constructed by
inversion of the 0.45-kb PvuII fragment containing part of the coding sequence of the lacZ fragment of pBluescript
KS(+). Insertion of the 5-kb PstI fragment which contains
the cloned mbgA gene into the PstI site of pGS240
yields plasmid pGS242. pGS246 was constructed by digestion of plasmid
pGS242 with SalI to delete a 2.3-kb fragment, followed by
religation. pGS281 was constructed by digestion of plasmid pGS242 with
EcoRV to delete a 0.8-kb fragment, followed by religation.
pGS249 was constructed by unidirectional deletion (6) with
exonuclease III of a 1.2-kb fragment downstream of the cloned
-galactosidase gene.
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FIG. 1.
Restriction map and genetic organization of the B. megaterium -galactosidase gene (mbgA) and
-galactosidase activities of deletion derivatives of the 5-kb
PstI fragment in E. coli. E, EcoRV; H,
HindIII; N, NaeI; P, PstI; S,
SalI.
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Sequence analysis revealed that
mbgA consists of 3,102 bp,
which corresponds to a protein monomer of 1,034 amino acids with
a
calculated molecular mass of 118,088 Da (data not shown). Comparison
of
the deduced amino acid sequence encoded by the
mbgA gene
with
those of the
-galactosidase of
E. coli
(
15),
Klebsiella pneumoniae (
3), and
Streptococcus thermophilus (
31) revealed 43.7, 45.4,
and 49.5% overall similarity, respectively. Immediately
downstream
of
mbgA is an inverted repeat with the sequence
AAGAA
GGACTTTCATT
TGAAAGTCCTTTTT,
which may serve as a putative
-independent transcription
terminator.
Further downstream is an incomplete ORF transcribed in the
opposite
direction (Fig.
1). Further upstream is an incomplete ORF
transcribed
in the direction opposite to that of the
mbgA
gene (Fig.
1). The
genetic organization of these ORFs suggests that
B. megaterium contains a
-galactosidase gene that exists
in a monocistronic
operon. This is unlike the case in many other
bacteria, where
the
-galactosidase gene and the lactose permease
gene usually
coexist in an operon (
15,
18,
26,
38). The
nucleotide
sequence of the
mbgA regulatory region is shown
in Fig.
2.
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FIG. 2.
Nucleotide sequence of the regulatory region of the
B. megaterium mbgA gene and base substitutions in CREs. (A)
The  A-like promoter sequence is overlined. The 27-bp
inverted repeat that overlaps the promoter sequence is indicated by a
pair of solid inverted arrows. Two partially overlapping CREs which are
contained in the 27-bp inverted repeat are designated CRE-I and CRE-II.
The N-terminal amino acids of ORF1 and mbgA are also shown.
The putative ribosome-binding sites (S/D) are boxed. (B)
pUBCAT-based plasmids pGS288, pGS320, pGS326, pGS340, and
pGS348 contain a cat reporter gene transcriptionally fused
to the mbgA promoter. pGS288 carries the wild-type
mbgA promoter, whereas pGS320, pGS326, pGS340, and pGS348
carry various base substitutions in CREs. The positions of the two
flanking primers, P207 (5'-GCGGGTCGACAAGCTCGCCTCTTCCTT-3')
and P208 (5'-GCGGAAGCTTTGGTAGTAAACAGA-3'), are also
indicated.
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Identification of the transcription initiation site of
mbgA.
To determine the precise transcription initiation site
of mbgA, RNA was isolated from log-phase B. megaterium cells harboring plasmid pGS288 (Fig. 2). A 16-mer
oligonucleotide (5'-CGTTTGCAGGTGCTGT-3') complementary to a
region extending from position +71 to position +86 relative to the
transcription start site of the mbgA gene was used as the
primer. Primer extension was carried out as described previously
(29), and the result is shown in Fig.
3. Only one major extension product was
detected, indicating that the 5' end of the mRNA for mbgA is
located 40 bp upstream of its translation start site (Fig. 2).
High-resolution S1 nuclease mapping (1) was also performed
to further confirm this result. Two major protected fragments with only
a one-base difference in length were observed (Fig. 3). One of them
corresponds to a transcription initiation site which is identical to
that identified by primer extension analysis. This transcription
initiation site is at an appropriate distance from a putative promoter
sequence (TTGATA for the 
35 box and TATCAT for
the 10 box) that may be recognized by the B. megaterium
equivalent of B. subtilis A (23).
A 27-bp inverted repeat overlaps the 35 and 10 regions of this
putative promoter (Fig. 2). Within the 27-bp inverted repeat are two
partially overlapping sequences (CRE-I and CRE-II) that exhibit
considerable sequence similarity to the CRE consensus sequence
(12).
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FIG. 3.
Determination of the transcription initiation site for
mbgA by primer extension analysis and S1 nuclease mapping.
(A) Primer extension analysis of transcripts was performed with RNA
from B. megaterium cells harboring pGS288. Lane 1 shows the
result of this analysis. (B) S1 nuclease mapping was carried out with
RNA isolated from the same source as that used for panel A. Lane 1 shows the protected products. For both panels, dideoxy sequencing
ladders obtained with the same primer used to make the probes for S1
nuclease mapping and primer extension experiments are shown. The
sequence indicated is complementary to that read from the ladder.
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Effects of lactose, glucose, and IPTG on -galactosidase
production in wild-type B. megaterium and the
mbgA mutant.
The effects of lactose, glucose, and IPTG
on -galactosidase production were examined by using B. megaterium cells grown in LB medium. -Galactosidase activities
were measured at 37°C with o-nitrophenyl--D-galactopyranoside (ONPG) as
the substrate by the method of Miller (20). It was found
that 2% lactose could increase -galactosidase production about
18-fold (Table 1). In the presence of 2%
lactose plus 2% glucose, about 25-fold repression exerted by glucose
was observed. Treatment with either 1 or 6 mM IPTG caused only a slight
increase in -galactosidase production compared to 2% lactose
treatment. These results suggest that -galactosidase production in
B. megaterium is lactose inducible and is subject to
catabolite repression by glucose. To examine the contribution of
mbgA to lactose-mediated induction of -galactosidase
production in B. megaterium, we disrupted the
mbgA gene in the B. megaterium chromosome by
using integrative plasmid pGS278, which was constructed as follows. A
600-bp EcoRI-HindIII fragment carrying an
internal sequence of the mbgA gene was generated by PCR
using synthetic oligonucleotides P194 (5'-GCCAAAGCTTAAATGGAAGC-3')
and P195 (5'-GCGGAATTCAGCTGAAACGCTAAGTT-3'). This
fragment was cloned into EcoRI- and
HindIII-cut plasmid pDG1515 (5) to get
plasmid pGS278. Disruption of the mbgA gene by a Campbell-like single-crossover recombination event was confirmed by
Southern blot analysis (29) (data not shown). The
mbgA mutant was then grown in LB medium in the absence or
presence of 2% lactose to an optical density at 600 nm of 0.6 for
-galactosidase activity assay. As shown in Table 1, disruption of
the mbgA gene in the B. megaterium chromosome
abolished the lactose-mediated induction of -galactosidase
production. This suggests that the mbgA gene is the only
lactose-inducible -galactosidase gene in B. megaterium.
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TABLE 1.
Effects of lactose, glucose, and IPTG on
-galactosidase production in wild-type B. megaterium and
an mbgA mutant
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Effects of mutations in CREs on catabolite repression of the
mbgA promoter-cat transcriptional fusions.
To assess the contributions of the two CRE-like sequences to catabolite
repression of -galactosidase production, we firstly introduced a DNA
fragment carrying the mbgA promoter and CRE-like sequences
into promoter probe vector pUBCAT (37). The
resulting plasmid, pGS288, bears an mbgA
promoter-cat transcriptional fusion (Fig. 2). B. megaterium cells transformed with plasmid pGS288 were grown in LB
medium in the absence or presence of lactose or lactose plus glucose.
Chloramphenicol acetyltransferase (CAT) activities were measured at
37°C by the spectrophotometric method of Shaw (32). It was
found that 2% glucose could still exert about ninefold catabolite
repression (Table 2).
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TABLE 2.
Effects of base substitutions in CREs on catabolite
repression of mbgA promoter-cat
transcriptional fusions
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We also made some
mbgA promoter-
cat
transcriptional fusion constructs in which CRE-I, CRE-II, or both were
mutated by the
two-step PCR method (
8) (Fig.
2). We then
examined the responses
of these fusions to lactose and glucose. For
each construct, at
least two independent clones were chosen for CAT
activity assays
in order to obtain consistent results. The results
showed that
mutations in either of the two CREs caused partial relief
of catabolite
repression (Table
2). Mutations in CRE-I of pGS320 and
CRE-II
of pGS326 decreased catabolite repression from about 9-fold in
the wild type to about 1.8-fold and 1.4-fold, respectively. These
results suggest that both CRE-I and CRE-II can function as an
active
CRE for catabolite repression of the
-galactosidase gene
of
B. megaterium. When CRE-I and CRE-II were mutated together
(pGS340
and pGS348 in Fig.
2), catabolite repression was still
not completely
abolished (Table
2). These results suggest that
either another
mechanism(s) is involved in catabolite repression
of
mbgA
expression or another CRE-like sequence(s) is located
in the coding
region of the
mbgA gene. Previous studies indicated
that, in
addition to a promoter-proximal CRE, a
cis-acting site
required for catabolite repression was also identified in the
coding
regions of the
hutP (
25,
39),
gntR
(
21,
22), and
xylA (
14) genes of
B. subtilis. We cannot exclude the possibility
that base
substitutions in the central CG or GG residues of the
two CREs could
only partially reduce the affinity of the catabolite
repressor for
these two sites. However, previous in vitro studies
indicated that
alterations of the central residues could prevent
binding of the
catabolite repressor to CREs identified in some
Bacillus
genes (
17,
22). It remains to be experimentally determined
whether two catabolite repressor molecules can bind to these two
CREs
simultaneously. Investigation by in vitro gel mobility shift
assays and
in vivo studies of the possible role of CcpA in catabolite
repression
of
mbgA expression via the two CREs should help to
clarify
these issues.
On the other hand, mutations of CRE-I, CRE-II, or both had differential
effects on basal expression from the
cat reporter
gene.
Mutations in CRE-I or pGS320 had little or no effect on
basal
expression, whereas mutations in CRE-II of pGS326 caused
about a
1.7-fold increase in the basal expression (Table
2).
The estimated
G values for the potential hairpin structure of
the 27-bp
inverted repeat (an 11-bp stem and a 5-base loop) in
pGS288, pGS320,
pGS326, pGS340, and pGS348 are
5.8,
3.6,
3.6,
9.8, and
3.6
kcal/mol, respectively, according to the method
of Tinoco et al.
(
34). Site-directed mutagenesis in pGS348 reduced
the dyad
symmetry (
G =
3.6 kcal/mol) of the 27-bp inverted
repeat,
whereas mutagenesis in pGS340 enhanced the dyad symmetry
(
G =
9.8 kcal/mol) in such a way that a perfect
27-bp inverted repeat
was obtained (Fig.
2). Mutations in both CRE-I
and CRE-II of pGS348
had little or no effect on the basal expression,
whereas mutations
in pGS340, which strengthen the hairpin, caused about
a 3.3-fold
reduction in the basal expression. Base substitutions in
either
half of the 27-bp inverted repeat did not directly change the
sequences in the
35 and
10 boxes of the
mbgA promoter.
However,
it was still possible that alterations of their flanking
sequences
might affect RNA polymerase recognition and binding, thus
altering
the promoter strength.
In contrast to the dramatic induction of chromosomal
-galactosidase
production by lactose,
cat expressions from the various
mbgA promoter-
cat transcriptional fusions on the
pUB110-derived
high-copy plasmids (
16) (ca. 50 copies per
chromosome) were
not lactose inducible (Table
2). One possible reason
is that
the concentration of the putative lactose-responsive repressor
(not the catabolite repressor) in
B. megaterium is
relatively
very low. A limited amount of the repressor might
efficiently
repress expression of the single-copy
mbgA gene
in the chromosome
(lactose inducible) but might not be enough to
repress expression
of multiple copies of the
mbgA gene on a
high-copy plasmid (not
lactose inducible). Another possibility is that
the lactose-responsive
cis-acting element is not contained
in the
mbgA promoter region
mentioned above.
In conclusion, we have identified two CRE-like sequences within the
27-bp inverted repeat which can exert catabolite repression
on
-galactosidase production in
B. megaterium. To our
knowledge,
this is the first example of two functional overlapping CREs
that
exist in the promoter region of a
Bacillus gene. In
CRE-II, the
central two residues are GG instead of the highly conserved
CG
residues. The sequencing markers shown in Fig.
3 clearly indicated
that this was not a sequencing error. Moreover, this is not
unprecedented;
it has been reported that a functional CRE identified in
the promoter
region of
gntR contains central TG residues
(
22). Although we
have demonstrated that the
-galactosidase of
B. megaterium is
capable of hydrolyzing
ONPG and X-Gal, the physiological substrates
for the
-galactosidase
of
B. megaterium remain undetermined.
Work using the
purified
-galactosidase of
B. megaterium is needed
to
determine if it can use lactose as a substrate.
Nucleotide sequence accession number.
The nucleotide sequence
reported here has been assigned GenBank accession no. AF047824.
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ACKNOWLEDGMENTS |
We are grateful to Douglas J. Cork, a Visiting Professor from the
Department of Biological, Chemical and Physical Sciences, Illinois
Institute of Technology, Chicago, for reviewing the manuscript.
This research was supported by grant NSC 86-2316-B-010-013-B19 from the
National Science Council of the Republic of China.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Biochemistry, School of Life Science, National Yang-Ming University,
Taipei, Taiwan 112, Republic of China. Phone: 886-2-2826-7127. Fax:
886-2-2826-4843. E-mail: gcshaw{at}mailsrv.ym.edu.tw.
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Journal of Bacteriology, September 1998, p. 4734-4738, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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