Previous Article
Journal of Bacteriology, February 1999, p. 1054-1058, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Two Roles for the Leucine-Responsive Regulatory Protein in
Expression of the Alanine Catabolic Operon (dadAB) in
Klebsiella aerogenes
Brian K.
Janes and
Robert A.
Bender*
Department of Biology, The University of
Michigan, Ann Arbor, Michigan 49109-1048
Received 11 September 1998/Accepted 16 November 1998
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ABSTRACT |
The lrp gene, which codes for the leucine-responsive
regulatory protein (Lrp), was cloned from Klebsiella
aerogenes W70. The DNA sequence was determined, and the clone was
used to create a disruption of the lrp gene. The lack of
functional Lrp led to an increased expression of the alanine catabolic
operon (dad) in the absence of the inducer
L-alanine but also to a decreased expression of the operon
in the presence of L-alanine. Thus, Lrp is both a repressor
and activator of dad expression. Lrp is also necessary for
glutamate synthase formation but not for the formation of two other
enzymes controlled by the nitrogen regulatory (Ntr) system, glutamate
dehydrogenase and histidase.
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TEXT |
Many bacteria use the amino acid
alanine as a carbon and nitrogen source by converting the
D-stereoisomer to pyruvate and ammonia. The two enzymes in
this catabolic pathway, alanine racemase and D-amino acid
dehydrogenase, have been studied for Klebsiella aerogenes,
Escherichia coli, and Salmonella typhimurium
(14, 15, 20, 26). The levels of both enzymes increase under
conditions of carbon limitation and when L-alanine is
present in the growth medium. In K. aerogenes enzyme levels
are increased by nitrogen limitation as well. The dad operon
(dadAB in S. typhimurium and K. aerogenes and dadAX in E. coli) contains a
gene that codes for one subunit of the dehydrogenase and another gene
that codes for the racemase. This operon is controlled by the
catabolite activator protein charged with cyclic AMP, the
leucine-responsive regulatory protein (Lrp), and, in the case of
K. aerogenes, the nitrogen assimilation control protein
(NAC) (15, 17, 19, 31). Catabolic activator protein-cyclic
AMP and NAC regulate dad by binding to the dad
promoter and increasing transcription of the operon. The regulatory
role of Lrp is more complex. Lrp is a repressor of dad in
E. coli, suggesting that the induction by alanine results
from overcoming the Lrp-mediated repression of dad
(19).
Lrp binds to the dad promoter of K. aerogenes in
vitro, but the addition of L-alanine or
L-leucine does not completely abolish the ability of Lrp to
bind (15). We therefore studied the role of Lrp in the
alanine-mediated induction of dad. The results presented here suggest two roles for Lrp in the alanine-dependent induction of
the dad operon, a role as a repressor and a role as an activator.
Cloning lrp from K. aerogenes W70.
In
order to clone the lrp gene from K. aerogenes W70
we took advantage of the fact that lrp from K. aerogenes RT48 had already been sequenced (12). Primers
that contained a recognition sequence for a restriction endonuclease
(EcoRI for the upstream primer and BamHI for the
downstream primer) and the 5'- or 3'-terminal 25 nucleotides of the
RT48 lrp gene were used to amplify a DNA fragment
approximately 500 bp in length from chromosomal DNA isolated from
KC2668 (which is derived from strain W70) by standard methods of PCR.
The exact conditions for the PCR have been described elsewhere (24). This fragment was cloned into the expression vector
pQE70, creating pCB1074, so that an
isopropyl-
-D-thiogalactopyranoside (IPTG)-inducible
promoter would drive the expression of any gene in the cloned fragment.
pCB1074 was introduced into an E. coli lrp mutant, BE2
(lrp-35; a complete list of strains and plasmids used in
this work is included in Table 1), and
tested for its ability to complement the lrp-35 allele. The
presence of pCB1074 restored two phenotypes of the lrp-35
allele to the wild-type state. lrp mutants grow slowly on
glucose minimal medium and are unable to use L-glycine as a
sole source of nitrogen (1, 16, 29). BE2 carrying pCB1074
grew well on glucose minimal medium and was able to use
L-glycine as the sole nitrogen source. It therefore
seemed likely that pCB1074 contained a functional lrp gene,
and it complemented the lrp-35 allele in the presence or absence of IPTG. DNA sequence analysis of the plasmid identified a gene
highly similar, but not 100% identical, to the lrp gene of
RT48. Thus, the gene isolated from W70 was different from the lrp gene of RT48, as well as from all other known
lrp sequences.
As a result of the cloning strategy, pCB1074 contains a hybrid
lrp gene. Most of it is from W70, but the 25 terminal
nucleotides
(both 5' and 3') are from RT48. In order to determine the
sequences
of the termini of the W70
lrp gene we cloned a
larger fragment
of DNA that contained the
lrp gene. Southern
blot analysis with
pCB1074 as a probe of chromosomal DNA from strain
KC2668 cut with
various restriction enzymes indicated that the
lrp gene would
be contained within a 5.5-kb
BamHI
fragment (data not shown).
Chromosomal DNA from KC2668 was digested
with
BamHI and cloned
into pUC19. The ligation mixture was
introduced directly into
EB4516 (created by moving the
lrp-35 allele into the restriction-deficient
strain YMC9)
and plated on glucose minimal medium with
L-glycine
as the
sole nitrogen source. A single colony which gave rise to
strain EB4517
was isolated after 4 days of incubation at 37°C.
This strain was
shown to contain a plasmid of the expected size
(pCB1075). When
introduced into strain DH5
and the
lrp mutant
EB4516,
plasmid pCB1075 caused the strains to grow poorly, and
in the latter
strain it did not allow growth with glycine as the
sole nitrogen
source. Subsequent DNA sequence analysis identified
an
lrp
gene in pCB1075 indistinguishable from that in pCB1074
(except for
three silent substitutions in the 3'-terminal 25 nucleotides).
Thus,
this
lrp gene appears to be fully functional. It has been
shown that high levels of Lrp are toxic to the cell (
5,
7).
Therefore, it seemed likely that pCB1075, which contained the
wild-type
lrp gene in a high copy number, was toxic to the
cell
and that the original host had developed an additional mutation
that compensated for the toxic effects of the high level of Lrp.
Inactivating the
lrp gene provided further evidence that the
overexpression
of
lrp was toxic. Plasmid pCB1076 was
constructed by cloning a
kanamycin resistance gene (from pWW-84) into a
unique
BglII site
contained in the
lrp gene. This
construct disrupted
lrp at codon
10, and therefore no
functional Lrp was provided by the plasmid.
Transformants of DH5
that contained this plasmid grew
normally.
Isolation of pCB1075 allowed us to obtain the complete sequence of the
lrp gene from W70. At the nucleotide level,
lrp
from
W70 is 95, 94, 94, 90, and 88% identical to
lrp from
RT48,
Enterobacter aerogenes,
Serratia
marcescens,
E. coli, and
S. typhimurium, respectively.
At the amino acid level, Lrp is
identical among these organisms
except at position 3 (glycine in
S. marcescens and serine in the
others) and at position
95 (alanine in
K. aerogenes W70 and
S. typhimurium, serine in
K. aerogenes RT48, and
threonine in the
others).
Isolation of an lrp mutant of K. aerogenes W70.
We isolated an lrp mutant of
K. aerogenes, KC4562, by reverse genetics
with the ampicillin-resistant suicide plasmid pAH34 (21). We cloned an internal fragment of the lrp
gene from pCB1075 into pAH34, resulting in pCB1093, and transferred
this plasmid into KC2668 by conjugation. Since pAH34 cannot replicate
in the absence of the pir gene, ampicillin-resistant
transconjugants were expected to result from the integration of the
plasmid into the chromosomal lrp gene. This integration
would result in a partially diploid strain with one lrp
allele that was 3' truncated at codon 104 (of 164) and another allele
that was 5' truncated at codon 10. The plasmid pAH34, containing the
bla gene, would separate the two nonfunctional alleles.
Ampicillin-resistant transconjugants were isolated after mating EB4594
with KC2668. PCR analysis of several of these integrants revealed that
the wild-type lrp gene was not present and that the pAH34
plasmid had integrated into the chromosome. Southern blot analysis also
revealed that the lrp gene had been disrupted (data not
shown). One of these strains was designated KC4562 (dadA1
lrp-101). Since the lrp-101 allele contains a
duplication of part of the lrp sequence, it should be
unstable and could revert to the wild type by RecA-mediated excision of
pCB1093. Therefore, we used consecutive P1 transductions to create
KC4602, a dad+ recA strain carrying the
lrp-101 allele. KC4602 (lrp-101) grew slowly on
minimal medium compared to KC3346 (lrp+). The
growth rates of these two strains were indistinguishable on minimal
medium supplemented with the branched-chain amino acids (leucine,
isoleucine, and valine [0.005% each]) and serine and 0.2% glutamine.
The addition of the branched-chain amino acids and serine had a
negative effect on the growth of KC4602 on minimal medium.
It was
necessary to supplement the medium with glutamine in order
to restore
wild-type growth. When the medium was supplemented
only with glutamine
the cells still grew at a lower rate than
did the wild type. Under
these conditions, the addition of branched-chain
amino acids and serine
was
beneficial.
Effect of lrp-101 on dad operon
expression.
The presence of L-alanine in the growth
medium leads to an induction of D-amino acid dehydrogenase
and alanine racemase. Levels as low as 2 mM have been used to study the
alanine-dependent induction of dad (19). We
suspected that these levels might not saturate the induction system,
and therefore we determined to what extent dad could be
expressed in the presence of L-alanine. Figure
1 shows that the dehydrogenase levels
continued to increase until L-alanine was present at levels
between 50 and 100 mM. We therefore chose to use 100 mM
L-alanine in our subsequent characterization of
lrp-101 to ensure that dad expression was
maximized. We have shown previously that L-alanine present
in the medium at 22 mM (0.2%) derepresses the nitrogen-regulatory
(Ntr) system by inhibiting glutamine synthetase (15). Then
the Ntr system activates dad expression through NAC. In
order to circumvent any role of NAC in the alanine-dependent activation
of dad in our study of lrp-101, we included 0.2%
L-glutamine in the medium. L-glutamine at these levels has been shown to prevent the derepression of the Ntr system by
alanine (15).
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FIG. 1.
Induction of D-amino acid dehydrogenase
(DAADH) by increasing amounts of L-alanine. Strain KC3346
was grown overnight in glucose-ammonium minimal medium and used to
inoculate fresh cultures that included L-alanine at
increasing concentrations. Cells were grown to mid-log phase and
assayed for DAADH as described previously (15). DAADH is
reported as specific activity (units per milligram of protein), and the
values are the means of three independent experiments.
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Table
2 shows the effect of the
lrp-101 allele on the expression of the
dad
operon and illustrates the two observed roles
for Lrp in the
alanine-dependent induction of
dad (although it
should be
noted that the two strains are not completely isogenic,
as KC4502 also
carries the
recA-3011 allele). In the absence of
alanine,
the level of dehydrogenase is four times higher in the
lrp
mutant than in the wild type. This suggests that Lrp is a
repressor of
dad expression in
K. aerogenes. However, in
the presence
of alanine, the level of dehydrogenase is four times lower
in
the mutant than in the wild type. This suggests that Lrp is an
activator (either direct or indirect) of
dad. The addition
of
alanine leads to a 25-fold increase of
dad expression in
the wild
type. While the addition of alanine has little or no effect on
dad expression in the mutant, the level of dehydrogenase is
much
higher than the repressed level of the wild type. Thus, it appears
that half of the alanine-dependent induction of
dad can be
explained
by the repression of the operon by Lrp, while the other half
requires
the activation of the operon by Lrp. This pattern repeats in
E. coli, although the evidence of Lrp as a repressor of
transcription
is not so clear (see results obtained for W3110 and BE2
without
alanine in Table
2).
Effect of lrp-101 on glutamate synthase
expression.
The lack of functional Lrp has effects on the
expression of enzymes other than those of alanine catabolism.
Glutamate synthase (the product of the gltBD operon) has
been well characterized as an Lrp-dependent operon in E. coli (5, 9, 10, 30). The levels of glutamate synthase
were reduced eightfold or more (Table 3)
in the K. aerogenes lrp-101 strain, consistent with the
observation that a functional Lrp is also necessary for the full
expression of gltBD in K. aerogenes.
Glutamate synthase plays a role in maintaining a functional Ntr system,
in that strains unable to produce functional glutamate synthase also
fail to derepress the Ntr system normally (26). However, any
role that the Ntr system plays in the expression of the enzymes studied
here has been circumvented by the addition of glutamine to the medium. Therefore, Lrp must play a direct role in the loss of glutamate synthase formation observed in the mutant. Other enzymes of the Ntr
regulon do not require functional Lrp for their formation, i.e.,
histidase and glutamate dehydrogenase (Table 3).
Conclusions and implications.
In this work we have
characterized an lrp mutant of K. aerogenes
W70. A strain that lacks functional Lrp grows poorly on minimal medium,
although the addition of glutamine, serine, and the branched-chain amino acids allows the strain to grow in a manner indistinguishable from that of the wild type. At the genetic level, Lrp is necessary to
activate gltBD and is both an activator and repressor of the dad operon. The role of Lrp in gltBD expression
and as a repressor of the dad operon is analogous to
well-studied systems in E. coli (6, 19, 23,
31). The growth phenotypes observed are similar to those seen in
E. coli lrp mutants, although to restore a wild-type growth
rate to K. aerogenes it was necessary to add glutamine to minimal medium containing branched-chain amino acids and serine. The
role of Lrp as an activator of the dad operon was unanticipated.
There are several possible explanations for how Lrp acts as both a
repressor and an activator of the
dad operon. The simplest
is that in the absence of alanine Lrp acts as a repressor of the
operon, but when alanine is bound to Lrp, a conformational change
occurs while Lrp is bound to the DNA that allows the protein to
activate transcription. Since Lrp has been shown to be either
an
activator or a repressor of several leucine-responsive operons,
and
alanine can mimic the effects seen with leucine, this model
does not
suggest a new function for Lrp (
6). There are several
Lrp
binding sites present in the
dad promoter, as is the case
for many Lrp-dependent operons (
6). Whether the hypothesized
conformational change resulting from alanine being bound to Lrp
would
result in different interactions between two or more Lrp
molecules,
between an Lrp complex and RNA polymerase, or between
Lrp molecules and
the DNA of the promoter is not
clear.
A recent study by Roesch and Blomfield (
28) has suggested
how Lrp could both activate and repress an operon. They show that
for
the
fim switch in
E. coli, a single Lrp binding
site in a
three-binding site complex is sensitive to leucine-bound Lrp.
In the absence of leucine, three Lrp molecules bind to the DNA
region
and recombination is inhibited. When leucine is present,
one
binding site can no longer keep Lrp bound, and recombination
is
stimulated by the remaining two Lrp molecules. It is possible
that a
similar interaction occurs at
dad in which an Lrp molecule
binding alanine can no longer bind to the DNA, and the absence
of Lrp
at this site is the cause of the derepression seen in
lrp mutants but not in the wild type. However, other Lrp molecules
bound to
the promoter do not exhibit this sensitivity and are
necessary to
activate
transcription.
Lrp could also be indirectly involved in the alanine-dependent
activation of
dad if there is a separate (but Lrp-dependent)
positive activator of
dad expression. In this scenario the
presence
of alanine would lead to the derepression of
dad by
releasing
the Lrp-mediated repression at the
dad promoter
and lead to the
activation of
dad through the proposed
Lrp-dependent activator.
However, this model requires the existence of
yet another regulator,
possibly the
dadQ allele identified
by others (
3,
11).
Other explanations for the dual role of Lrp in the alanine-dependent
activation of
dad, including Lrp-dependent alanine transport
and Lrp's effect on the gene that codes for the other dehydrogenase
subunit, are possible but seem even less likely. At least two
systems
have been identified that transport alanine in
E. coli,
the
cycA system and the LIV-1 system (
8,
20,
25,
27).
It is not known whether Lrp plays a role in the
cycA system,
but
the lack of functional Lrp leads to higher levels of expression
of
the LIV-1 system (
13). Therefore, transport of alanine via
LIV-1 should not be reduced in an
lrp mutant strain. In
addition,
KC4602 can use alanine as its sole nitrogen source, although
its
growth rate under this condition is severely reduced compared
to
that of the wild type (data not shown). Although we have no
data to
address the regulation of the gene that codes for the
other
dehydrogenase subunit, the pattern of expression for alanine
racemase
mimics that of the dehydrogenase (data not shown). Therefore,
no matter
how the other gene is regulated, Lrp plays two roles
in regulating
dadAB.
Our results lead us to favor the idea that the
dad promoter
can be in three states with respect to Lrp: it can be free of
Lrp or it
can be occupied by inducer-free Lrp or inducer-bound
Lrp. Inducer-free
Lrp represses transcription below basal level,
the absence of Lrp leads
to basal-level expression, and inducer-bound
Lrp leads to activation of
transcription above basal level. These
three hypothetical states and
the existence of two possible inducers
(leucine and alanine) suggest
that the role played by Lrp in genetic
expression may be quite complex.
This complexity is reflected
in the large number of cellular responses
in which Lrp plays a
role.
Nucleotide sequence accession number.
The DNA sequence of
lrp from K. aerogenes W70 has been submitted
to GenBank and has been assigned accession no. AF090144.
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ACKNOWLEDGMENTS |
This work was supported by Public Health Service grant GM 47156 from the National Institutes of Health to R.A.B.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology, The University of Michigan, Ann Arbor, MI 49109-1048. Phone: (734) 936-2530. Fax: (734) 647-0884. E-mail:
rbender{at}umich.edu.
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Journal of Bacteriology, February 1999, p. 1054-1058, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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