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Journal of Bacteriology, May 1999, p. 2889-2894, Vol. 181, No. 9
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
In Vitro Transcriptional Studies of the
bkd Operon of Pseudomonas putida:
L-Branched-Chain Amino Acids and D-Leucine
Are the Inducers
Kunapuli T.
Madhusudhan,
Jinhe
Luo, and
John R.
Sokatch*
Department of Biochemistry and Molecular
Biology, University of Oklahoma Health Sciences Center, Oklahoma
City, Oklahoma 73190
Received 30 November 1998/Accepted 6 February 1999
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ABSTRACT |
BkdR is the transcriptional activator of the bkd
operon, which encodes the four proteins of the branched-chain keto acid
dehydrogenase multienzyme complex of Pseudomonas putida. In
this study, hydroxyl radical footprinting revealed that BkdR
bound to only one face of DNA over the same region identified in
DNase I protection assays. Deletions of even a few
bases in the 5' region of the BkdR-binding site greatly
reduced transcription, confirming that the entire protected region is necessary for transcription. In vitro transcription of the bkd operon was obtained by using a vector
containing the bkdR-bkdA1 intergenic region plus the
putative 
-independent terminator of the bkd
operon. Substrate DNA, BkdR, and any of the
L-branched-chain amino acids or D-leucine was
required for transcription. Branched-chain keto acids,
D-valine, and D-isoleucine did not promote
transcription. Therefore, the L-branched-chain amino acids
and D-leucine are the inducers of the bkd
operon. The concentration of L-valine required for
half-maximal transcription was 2.8 mM, which is similar to that needed
to cause half-maximal proteolysis due to a conformational change in
BkdR. A model for transcriptional activation of the bkd
operon by BkdR during enzyme induction which incorporates these
results is presented.
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INTRODUCTION |
Pseudomonas putida
biotypes (20) are major players in the bioremediation of
soil and water and metabolize a wide variety of natural and human-made
compounds. P. putida possesses inducible pathways for
the oxidation of many, if not all, amino acids even though they
are required for protein synthesis. Branched-chain keto acid
dehydrogenase is a multienzyme complex which catalyzes the oxidative
decarboxylation of branched-chain keto acids resulting from
transamination of L-branched-chain amino acids or oxidation of D-branched-chain amino acids. There is a good deal
of interest in the structure and function of keto acid dehydrogenase
multienzyme complexes (12) because of their tremendous size
and because mutations in humans affecting the components of the complex
result in serious genetic diseases (15).
The four proteins of the complex are encoded by the structural genes of
the bkd operon, the expression of which is under
positive control by BkdR (10). BkdR is a homologue of Lrp,
which is a global transcriptional regulator in Escherichia
coli (2). Lrp (for leucine-responsive protein) can act
as an activator or a repressor in the presence of leucine or may be
unaffected by the presence of L-leucine. In contrast, the
only known function of BkdR is to activate transcription of the
bkd operon. Expression of the bkd
operon of P. putida is induced by growth on
branched-chain amino or keto acids (11) and subject to
catabolite repression by glucose, succinate, or ammonium ion
(21). Branched-chain keto acid dehydrogenase is induced in
media containing D- or L-branched-chain amino acids or branched-chain 
-keto acids (11).
Since BkdR is a transcriptional activator of the bkd
operon, one would predict that the actual inducers would be
required for BkdR-mediated transcription. Previous data implicated
L-branched-chain amino acids as the inducers because
addition of L-branched-chain amino acids to DNase I
protection assays enhanced the appearance of hypersensitive sites in
the protected region (9). However,
L-branched-chain amino acids did not affect either the
stoichiometry of the BkdR-DNA complex or its mobility (5).
D-Valine and -ketoisovalerate had no effect on the
protection pattern. Enhancement of hypersensitive sites suggested that
L-branched-chain amino acids effected a conformational change in BkdR, which was subsequently demonstrated by circular dichroism spectroscopy and limited proteolysis (8). Although L-branched-chain amino acids did not affect the migration
of BkdR-DNA complexes in gel shift assays, they did affect bending of
DNA caused by binding of BkdR (6); BkdR alone caused a bend
angle of 92° while the angle formed by binding of BkdR plus
L-valine was 76°.
These data predicted that L-valine and probably other
L-branched-chain amino acids were the actual inducers of
the bkd operon. The objective of this study was to
identify the true inducers directly by determining the requirements for
in vitro transcription of the bkd operon and to
obtain additional information about the binding of BkdR.
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MATERIALS AND METHODS |
Bacterial strains and media.
For induction of branched-chain
keto acid dehydrogenase, P. putida PpG2 was grown in
0.3% L-valine and 0.1% L-isoleucine mineral salt medium (11) with aeration at 30°C. This strain grows
more rapidly in valine-isoleucine medium than in medium with
L-valine alone because L-valine inhibits
isoleucine synthesis. Mutants affected in bkdR or the
bkd operon cannot metabolize branched-chain amino
acids and are grown in valine-isoleucine medium plus a nonrepressing metabolizable substrate such as lactate or gluconate. E. coli DH5 was grown in 2× YT (18) medium with 200 µg of ampicillin/ml when carrying pUC plasmids. When P. putida carried pKRZ-1 plasmids, it was grown in medium with 90 µg of kanamycin/ml.
Reagents.
BkdR was prepared as described in reference
9. The samples of D-leucine were
obtained from TCI and Aldrich Chemical Company. E. coli RNA
polymerase holoenzyme, saturated with 
70, was obtained
from Epicentre Technologies.
Plasmid constructions.
The plasmids used in this study are
summarized in Table 1. pJRS168 was the
template used for in vitro transcription of the bkd
operon. For construction of pJRS168, an
EcoRV/SalI fragment was transcloned from pJRS25
(7) to pBluescript II SK(+). This fragment contains
nucleotides (nt) 1407 to 1753 (Fig. 1)
and includes nt 1544, the transcriptional start site of the
bkd operon. The EcoRV/SalI
fragment was then transcloned into pJRS85, with EcoRI and
KpnI pJRS85 contains the -independent transcriptional
terminator of the bkd operon on a
KpnI/SphI fragment (nt 6497 to 6733 [GenBank sequence M57613]) in pUC19. This placed the terminator immediately downstream of the nucleotides encoding the transcript. The host for pJRS168 was E. coli DH5 (Gibco
BRL), and DNA was purified with a QIAprep Spin Miniprep kit (Qiagen).
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FIG. 1.
Nucleotide sequence of the DNA fragment studied in this
report. Nucleotide numbering is taken from GenBank sequence M57613. The
shaded bases are those protected from the action of hydroxyl
radicals by binding of BkdR. Part of the coding sequences for
bkdR and bkdA1 are translated to show the
location of these genes. The arrows indicate the 5' ends of the DNA
fragments summarized in Table 2. The bend center is marked by the
inverted triangle, and the transcriptional start is marked +1. There is
no identifiable  35 hexamer.
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Plasmids pJRS177 to -179 and pJRS185 to -187 were constructed from the
same PCR fragments but were in different vectors. The
vector for
pJRS177, -178, and -179 was pKRZ-1 (
17), while the
vector
for pJRS185, -186, and -187 was pUC19 (
25). The fragments
were created by PCR with pJRS82 (
4) as the template as
described
in reference
6. The forward primers were
141, 142, and 142,
and the reverse primer was 87 in all cases (Table
2). The PCR
fragments were blunted with
Klenow reagent and deoxynucleoside
triphosphates and then cloned into
the
EcoRV site of pBluescript
SK(+) (Stratagene). The
fragments were released from pBluescript
SK(+) with
XbaI and
HincII and cloned into the
XbaI and
SmaI sites
of pKRZ-1, creating pJRS177, -178, and -179. Again, the host for
these plasmids was
E. coli DH5
, and
the plasmids were introduced
into
P. putida PpG2 by
triparental mating (
19). For the data
shown in Table
3,
P. putida PpG2
containing pJRS177 to -179 was
grown in valine-isoleucine medium to an
A660 of 0.5, harvested,
disrupted by sonic
oscillation, centrifuged at 90,000 ×
g for
1 h,
and then assayed for
-galactosidase (
14).
For the construction of pJRS185, -186, and -187, PCR fragments
described in the previous paragraph were transcloned from the
pBluescript SK(+) intermediate constructs into pUC19. The PCR
fragments
were released from the pBluescript intermediate constructs
by digestion
with
EcoRI and
XhoI and transcloned into pJRS168,
also digested with
EcoRI and
XhoI. By this means,
the insert of
pJRS168 was replaced with the PCR fragments cloned into
the pBluescript
intermediate constructs. The host for pJRS185 to -187 was
E. coli DH5
, and DNA was purified with a QIAprep Spin
Miniprep kit
(Qiagen).
DNase I and hydroxyl radical footprinting.
DNase I
footprinting was performed as described in an earlier study
(8). Hydroxyl radical footprinting was carried out as
described in reference 22. Fragments A and B,
previously used for DNase I footprinting (8), were also used
for hydroxyl radical footprinting. Fragment A included nt 1300 to 1678 (Fig. 1), and fragment B included nt 1407 to 1678 (Fig. 1). Fragment A
was labeled with [-32P]dCTP and Klenow reagent at the
3' end of the bottom strand shown in Fig. 1, and fragment B was labeled
at the 3' end of the top strand shown in Fig. 1 by the same procedure.
Radioactivity was measured with a Molecular Dynamics phosphorimager.
In vitro transcription.
The volume of the reaction mixture
was 35 µl. The buffer contained (final concentrations) 100 mM
potassium glutamate, 50 mM KCl, 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA,
10 µg of bovine serum albumin/ml, 1 mM CaCl2, 5%
glycerol, 1 mM dithiothreitol, and 5 mM MgCl2. Next, DNA
template was added and the mixture was incubated for 5 min. Then
1.2 × 10
9 M RNA polymerase 70
holoenzyme (Epicentre Technologies); 250 µM (each) ATP, GTP, and CTP;
25 µM [-32P]UTP; and 50 µg of heparin per ml were
added, and the mixture was incubated for an additional 5 min at 37°C.
The reaction was chased with 250 µM UTP; incubated for 10 min; and
then terminated with 2 volumes of ethanol, a 1/3 volume of 3 M sodium
acetate, and 2 µl of 1% yeast tRNA as a carrier for the
precipitation of RNA. The precipitate was dissolved in 6 µl of
formamide dye mixture (75% formamide, 0.075% xylene cyanol, and
0.075% bromphenol blue) and then resolved in an 8% urea-6%
polyacrylamide gel. The amount of radioactivity in the gel was
determined by use of the phosphorimager.
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RESULTS |
Hydroxyl radical footprinting.
The footprint of BkdR on its
substrate DNA, which was protected from the action of DNase I
(9), extended from approximately nt 1420 to nt 1520 (Fig.
1). Addition of L-valine resulted in formation of
hypersensitive sites at about nt 1453, 1475, and 1495 on the top strand
and about nt 1455 and 1495 on the bottom strand. Hydroxyl radical
footprinting was used to further characterize the binding of BkdR
to substrate DNA (Fig. 2). Simultaneous
chemical sequencing of DNA (13) identified specific
nucleotides that were protected from hydroxyl radicals by binding of
BkdR. The region of DNA covered by the hydroxyl radical footprint
is nearly identical to that covered by the DNase I footprint
(9). An interesting feature of the hydroxyl radical
footprint is that protection is phased at about every 10 bases on
both the top and bottom strands, which indicates that BkdR binds to one
face of DNA (Fig. 1).
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FIG. 2.
Hydroxyl radical footprint of the BkdR-DNA complex.
(A) Sequence of the top strand in Fig. 1. (B) Sequence of the bottom
strand in Fig. 1.
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Upstream boundary of the region required for transcription.
In
a previous study (6), it was shown that the downstream
boundary of the region required for transcription of the bkd operon was very close to the transcriptional start site of the bkd operon (nt 1544 [Fig. 1]). In order to
determine the upstream boundary of this region, several PCR-amplified
fragments were constructed with 5' ends at nt 1418, 1434, and 1449 and
the 3' end at nt 1698 (Fig. 1 and Materials and Methods). These
fragments were cloned into pKRZ-1, with the bkd promoter
oriented toward the promoterless lacZ reporter gene
(17). The host for these plasmids was P. putida PpG2 grown in valine-isoleucine (inducing) medium as
described in Materials and Methods. The results of the -galactosidase assays are shown in Table 3. There was a
noticeable decrease in the rate of transcription when the upstream
nucleotide pair was 1434, which is just inside of the
BkdR-binding region (Fig. 1). There was a much greater decrease, with
only ~4% of the activity retained, when the upstream boundary was nt
1449, which is near the bend center of the transcriptionally active DNA
fragment (Fig. 1) (6) and in a region of six consecutive thymines. Therefore, the DNA fragment necessary for transcription of
the bkd operon in vivo extends from the start of the
BkdR-binding region to the transcriptional start site of the
bkd operon.
In vitro transcription of the bkd operon.
The vector constructed for in vitro transcription studies of the
bkd operon was pJRS168, which contains the
entire region to which BkdR binds, fragments of bkdR
and bkdA1, and a transcriptional terminator in pUC19. The
terminator was the putative -independent terminator of the
bkd operon located downstream of lpdV,
the final gene of the operon (1). The length of the
transcript was calculated to be 368 bp, including restriction sites
introduced during cloning.
The factors required for transcription of the
bkd
operon were identified by the experiment whose results are
shown in Fig.
3. Template DNA in the form
of pJRS168, RNA polymerase containing
70, BkdR, and
L-valine all were required for transcription. There
was
very little expression of the
bkd operon by RNA
polymerase
in the absence of BkdR, or RNA polymerase plus BkdR, but
transcription
was increased at least 8- to 10-fold over the background
by the
addition of
L-valine. Therefore,
L-valine is one of the inducers
of the
bkd
operon. Some transcript was obtained with linear DNA,
but
transcription was much more efficient with pJRS168, which
provided
supercoiled DNA (data not shown). Since RNA polymerase
used in these
experiments was
70 holoenzyme and since there is a
10
hexamer, TAAGAT, just upstream
of the transcriptional start site of the
bkd operon (nt 1544 [Fig.
1]), it can be concluded
that the
bkd operon is transcribed from
a
70 promoter. Because the transcript was the correct
size, it can
also be concluded that the presumed
-independent
terminator functioned
as predicted. The fragment of DNA protected from
the action of
DNase I (
9) and hydroxyl radicals (Fig.
1) by
BkdR and that
is required for the expression of
bkd
operon-
lacZ fusions (Table
3) is about 90 bp, which
is unusually long. However, in vitro
transcription experiments
confirmed that this large fragment was
necessary for transcription. A
series of plasmids was constructed
with the same PCR fragments as those
shown in Table
3 plus the
downstream
-independent terminator and
used for in vitro transcription
(Fig.
4).
Very little transcript was obtained when the upstream
end of the
fragment was nt 1449, which is in the BkdR-binding
region near the bend
center (Fig.
1). When the upstream end of
the PCR fragment was nt 1434 or 1418, the amount of transcript
was similar to that obtained with
pJRS168.
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FIG. 3.
Factors required for in vitro transcription of the
bkd operon. The DNA template was 1.8 × 1010 M pJRS168, and the concentration of
L-valine was 10 mM. The protocol and the concentrations of
the other reactants are described in Materials and Methods. The arrow
indicates the expected size of the message. RNAP, RNA polymerase.
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FIG. 4.
Effect of deletions at the 5' end of the BkdR-binding
region on in vitro transcription. Because the PCR fragments were
slightly shorter than the fragment in pJRS168, the size of the
transcript was smaller, 321 compared with 368 bp for pJRS168. The
arrows indicate the size of the expected transcript. WT, wild type.
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Half-maximum transcription was obtained with a BkdR tetramer
concentration of 2.5 × 10
9 M with pJRS168 as the
DNA template. The calculated
Km for
L-valine
in substrate saturation studies of transcription
was 2.8 mM. The
saturation curve was conventional, although there was
some inhibition
of transcription above 25 mM
L-valine.
Binding of
L-valine causes
a conformational change in BkdR,
which enhances proteolysis of
BkdR by trypsin (
8). The
half-maximal velocity of proteolysis
was obtained at 2.5 mM
L-valine, which is very close to the figure
obtained in
this study for in vitro
transcription.
Other ligands which promote transcription.
The L
and D forms of valine, leucine, isoleucine, and
-ketoisovalerate were tested at 15 mM for their ability to promote transcription with pJRS168 as the DNA template (Fig.
5). The L forms of all three
branched-chain amino acids stimulated transcription; therefore, L-leucine and L-isoleucine
are inducers of the bkd operon in addition to
L-valine. -Ketoisovalerate and the D forms of valine and isoleucine had no effect and can be ruled out as inducers, even though branched-chain keto acid dehydrogenase is induced
by growth in media with these compounds. Surprisingly, the
D form of leucine was nearly as effective as
L-leucine in stimulating transcription, a result that
was obtained with D-leucine from two different suppliers.
The Km for L-leucine was 4.4 mM, and the Km for D-leucine was 6.2 mM.
The optical rotation obtained from both bottles of
D-leucine was the same as the literature value, and so the
bottles were not mislabeled.
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FIG. 5.
Ability of L-branched-chain amino acids and
D-leucine to initiate in vitro transcription of the
bkd operon. The DNA template was pJRS168, and the
potential ligands were tested at 15 mM. The arrow indicates the size of
the expected transcript. kiv, ketoisovalerate.
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Effect of D-leucine on DNase I footprint.
The
addition of L-valine to BkdR and substrate DNA caused an
enhancement of the hypersensitive sites when treated with DNase I
(9). In view of the activity of D-leucine in
transcription, and the failure of D-valine and
D-isoleucine to stimulate transcription, the DNase I
footprint obtained in the presence of D-leucine was compared with that obtained with L-valine (Fig.
6). The footprints were identical, so
that the effect of D-leucine on transcription must be the
same as that of the L-branched-chain amino acids and not an
artifact. Therefore, D-leucine must also be an inducer of
the bkd operon.
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FIG. 6.
Effect of D-leucine on the DNase I
protection assay. (A) Pattern obtained with the top strand in Fig. 1.
(B) Pattern obtained with the bottom strand. Each of the ligands was
tested at 15 mM, and the labeled fragments were the A and B fragments
used in reference 9.
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DISCUSSION |
Transcription of the bkd operon during
induction.
In noninducing media, BkdR binds to substrate DNA
(5) but transcription does not take place because BkdR and
DNA are not in the correct configuration. Transcriptionally inactive
BkdR binds to one face of a 90-bp segment of DNA which extends from the
initiating codon of bkdR to 25 relative to the
transcriptional start site of the bkd operon (Fig.
1). When P. putida grows in an inducing medium, the
intracellular concentration of L-branched-chain amino acids
(or D-leucine) reaches the near-millimolar value, and
several changes occur, the net result of which is transcription of the
bkd operon. Induction obtained when P. putida is grown in media with D-valine,
D-isoleucine, or branched-chain keto acids must be due to
the conversion of these compounds to L-branched-chain amino
acids (11). The concentration of L-valine which
results in half-maximal transcription is high enough to prevent
induction of branched-chain keto acid dehydrogenase in the absence of
exogenous branched-chain amino acids. Conversely, the concentration of
L-branched-chain amino acids in the pool is not high enough
to cause induction.
The presence of inducers brings about two important changes
that are essential for transcription of the
bkd
operon; BkdR undergoes
a conformational change to active BkdR
(
8) and substrate DNA
is bent to the correct angle
(
6). The conformational change
exposes the hinge region of
BkdR between the DNA-binding domain
and the rest of BkdR, making it
susceptible to proteolysis (
8).
The BkdR and RNA
polymerase-binding regions are very close together,
and so they can
easily come in contact. However, the change in
bend angle of DNA and
change in conformation of BkdR are necessary
to bring the
transcriptional complex into the correct configuration
for
transcription.
The BkdR DNA-binding site.
BkdR binds one face of a long
stretch of DNA between nt 1430 and 1520 (Fig. 1) (6). Lrp
binds to one face of an Lrp-binding site in the leader region of the
ilvGMEDA operon (16), which is discussed
below. It is interesting that the moles percent guanine plus cytosine
for the BkdR-binding site is 42% compared to 64% for the GenBank
sequence M57613 which includes bkdR, the intergenic region,
and the structural genes of the bkd operon of
P. putida. The BkdR DNA-binding site very likely
overlaps the transcriptional start site for bkdR, which
would explain the repression of bkdR expression by BkdR
(9). This is a rather large DNA-binding site, but since the
stoichiometry of BkdR tetramer per mole of substrate DNA is 3:1
(5) and the Stokes radius is 32 Å (8), a large
segment of DNA would be required. Binding of BkdR is probably cooperative since there was only one identifiable BkdR-DNA complex seen
in gel shift assays (9), and cleavage of the binding site near the center of the bend angle resulted in total abolition of DNA
binding (8). Inspection of the bases protected from hydroxyl
radicals (Fig. 1) did not reveal any consistent BkdR-binding motif.
There is a difference between in vivo transcription and translation and
in vitro transcription since less
-galactosidase
than transcript was
produced when the 5' end of the DNA fragment
was nt 1434 (Table
3 and
Fig.
4). For some unknown reason, the
entire in vivo transcript is not
converted to translatable message.
However, the two systems are not
directly comparable, and there
are many possible explanations for this
result. For example, the
message obtained from the
lacZ
fusion is not a normal message,
and this transcript may not be as
stable as the normal
transcript.
Comparison of Lrp and BkdR.
Since BkdR and Lrp are
homologous transcription factors, it is interesting to compare their
properties and activities. The major functional difference is that Lrp
is a global regulator while BkdR is a specific activator of the
bkd operon. This is reflected in a 100-fold
difference in copy numbers per cell: ca. 3,000 for Lrp (2)
and 25 to 40 for BkdR (9). It is interesting, however, that
Lrp can complement bkdR mutations in P. putida (10). Lrp can be either an activator or a
repressor, and leucine may or may not have an effect on the action of
Lrp (2). BkdR is strictly a transcriptional activator of the
bkd operon, which requires
L-branched-chain amino acids or D-leucine for
expression. There is 36.5% sequence identity between Lrp and BkdR and
58% similarity. An interesting difference between the two proteins is
that the pI for Lrp is 9.24 (24) and the pI for BkdR is 5.89 (10). Lrp is a dimer (24), while BkdR is a
tetramer (8).
Lrp produces several complexes with DNA (
3), while BkdR
produces a single complex with a stoichiometry of three BkdR tetramers
to one of substrate DNA (
5). There are multiple DNA-binding
sites for Lrp on the chromosome of
E. coli (
16,
23) but only
one binding site for BkdR on the chromosome of
P. putida (
10).
A consensus binding site for
Lrp was reported in reference
3 as YAGHAWATTWT where
Y = C or T, H = not G, W = A or T, D = not
C, and
R = A or G. Rhee et al. (
16) found a high-affinity,
14-bp
Lrp consensus sequence in the leader region of the
ilvGMEDA operon
of
E. coli. Inspection of
the binding region for BkdR (Fig.
1)
did not reveal a clear Lrp
consensus-binding site; however, there
are several regions which
are rich in A and T. Rhee et al. (
16)
also reported
that Lrp bound to one face of the primary and secondary
Lrp-binding
sites, which is the case with BkdR as reported in
this study
(Fig.
1 and
2).
| |
ACKNOWLEDGMENTS |
This research was supported by Public Health Service grant DK
21737 and Presbyterian Health Foundation grant C5142801.
We extend our appreciation to Fritz Schmitz for the optical rotation analyses.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Molecular Biology, University of Oklahoma Health
Sciences Center, P.O. Box 26901, Oklahoma City, OK 73190. Phone:
(405) 271-2227. Fax: (405) 271-3092. E-mail:
john-sokatch{at}ouhsc.edu.
| |
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Journal of Bacteriology, May 1999, p. 2889-2894, Vol. 181, No. 9
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
