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Journal of Bacteriology, June 2008, p. 4351-4359, Vol. 190, No. 12
0021-9193/08/$08.00+0     doi:10.1128/JB.00295-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The ArgP Protein Stimulates the Klebsiella pneumoniae gdhA Promoter in a Lysine-Sensitive Manner{triangledown}

Thomas J. Goss*

Department of Molecular, Cellular and Developmental Biology, The University of Michigan, Ann Arbor, Michigan 48109-1048

Received 27 February 2008/ Accepted 5 April 2008


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ABSTRACT
 
The lysine-sensitive factor that binds to the upstream region of the Klebsiella pneumoniae gdhA promoter and stimulates gdhA transcription during growth in minimal medium has been proposed to be the K. pneumoniae ArgP protein (M. R. Nandineni, R. S. Laishram, and J. Gowrishankar, J. Bacteriol. 186:6391-6399, 2004). A knockout mutation of the K. pneumoniae argP gene was generated and used to assess the roles of exogenous lysine and argP in the regulation of the gdhA promoter. Disruption of argP reduced the strength and the lysine-dependent regulation of the gdhA promoter. Electrophoretic mobility shift assays using crude extracts prepared from wild-type and argP-defective strains indicted the presence of an argP-dependent factor whose ability to bind the gdhA promoter was lysine sensitive. DNase I footprinting studies using purified K. pneumoniae ArgP protein indicated that ArgP bound the region that lies approximately 50 to 100 base pairs upstream of the gdhA transcription start site in a manner that was sensitive to the presence of lysine. Substitutions within the region bound by ArgP affected the binding of ArgP to the gdhA promoter region in vitro and the argP-dependent stimulation of the gdhA promoter in vivo. These observations suggest that elevated intracellular levels of lysine reduce the affinity of ArgP for its binding site at the gdhA promoter, preventing ArgP from binding to and stimulating transcription from the promoter in vivo.


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INTRODUCTION
 
In enteric bacteria, L-glutamate serves important roles as the primary donor of amino groups in the biosynthesis of other nitrogenous compounds (13) and as the potassium counterion in osmotic homeostasis (6). Enteric bacteria synthesize glutamate either by the pathway mediated by glutamine synthetase (GS) and glutamine, 2-oxoglutarate amidotransferase (GOGAT) or by the pathway mediated by only glutamate dehydrogenase (GDH) (for reviews, see references 21 and 28). In the absence of a functional GS-GOGAT pathway, the rate of glutamate biosynthesis by the GDH pathway alone may restrict growth under nitrogen-limited or hyperosmotic conditions (7, 18). Although the mechanism regulating the expression of GS in enteric bacteria has been studied extensively, those regulating GOGAT and GDH remain poorly understood (21).

In general, GDH activities in cells grown in minimal medium are tenfold higher than those in cells grown in amino acid-rich medium (3, 4). Although the factors contributing to this tenfold difference are unknown, several studies suggest that supplements such as casamino acids, glutamate, or other amino acids to minimal medium may each repress GDH expression as much as threefold (21). The activity of GDH in minimal medium-grown Klebsiella pneumoniae is reduced twofold in response to supplementation of the medium with L-lysine HCl to 0.01% (wt/vol) (550 µM) (10). This lysine-dependent repression of GDH acts at the transcriptional level (10). The lysine-dependent repression of GDH activity may contribute to the differential expression of GDH during growth in minimal and amino acid-rich media.

Another pattern of GDH expression that is inconsistently seen among enteric bacteria occurs during nitrogen-limited growth in minimal medium. In K. pneumoniae, nitrogen-limited growth results in tenfold or greater repression of the gdhA promoter, gdhAp, by the nitrogen assimilation control (NAC) protein (21). NAC also weakly represses the K. pneumoniae gdhA promoter two- to threefold by a second mechanism that requires only the upstream NAC binding site centered at –89 and is independent of the ability of NAC dimers to tetramerize (8, 22). This weaker NAC-mediated repression of gdhAp is abolished when the growth medium is supplemented with 550 µM lysine or when gdhAp carries either the A(–96)T substitution or the {Delta}–82 deletion that removes part of the NAC binding site centered at –87 (8, 22). In addition, the lysine-dependent regulation of gdhAp is essentially abolished when gdhAp carries either the A(–96)T substitution or the {Delta}–82 deletion (8). The similar effects that these modifications to gdhAp have on the weak NAC-mediated repression and the lysine-dependent repression suggest that these repression mechanisms act through a common mechanism to reduce gdhAp activity two- to threefold. Thus, it was proposed that exogenous lysine and NAC interfere with the ability of a putative transcriptional activator to stimulate transcription from gdhAp during growth in minimal medium. Although the mechanism by which exogenous lysine interferes with this stimulation was unclear, the data suggested that the weaker NAC-mediated repression of gdhAp results from the mutually exclusive binding of NAC and this putative lysine-sensitive transcriptional activator to overlapping binding sites in the upstream region of gdhAp (8, 22).

The identity of the putative lysine-sensitive transcriptional activator remained obscure until it was reported that either exogenous lysine or the disruption of the argP gene led to an osmosensitive phenotype in GOGAT-deficient Escherichia coli strains (18). This phenotype results from the inability of these strains to up-regulate GDH expression and increase intracellular glutamate levels under hyperosmotic growth conditions. As ArgP, the product of the argP gene, is known to be a DNA-binding transcription factor (17), it was speculated that ArgP stimulated the E. coli and K. pneumoniae gdhA promoters in a manner analogous to the lysine-sensitive activation of the E. coli argO promoter, argOp, by ArgP (17, 18). In addition, purified E. coli ArgP was shown to bind the upstream region of the E. coli argO promoter and either prevent transcription in the presence of lysine or activate transcription in the presence of either L-arginine or its analog L-canavanine (11).

NAC and ArgP are members of the LysR-type transcriptional regulator (LTTR) family of proteins, all of which display a significant level of amino acid sequence homology (24). Studies of the LTTR-dependent regulation of target promoters suggest that many of these proteins use a common mechanism to activate their target promoters (20 and see reference 24 for a recent description). Basically, this emerging paradigm postulates that the LTTR interacts with two closely spaced, dissimilar binding sites that lie in the regions upstream of target promoters. The promoter-distal binding site, often referred to as the recognition binding site (RBS), contains the centrally located LTTR consensus binding motif (5'-T-N11-A-3') and provides interactions that are required to anchor the LTTR to the upstream region of the target promoter. The LTTR binds to the RBS regardless of the presence of the effector, a low-molecular-weight molecule that binds to and alters the conformation of the LTTR (20, 24). The promoter-proximal binding site, referred to as the activation binding site (ABS), provides secondary interactions required for the LTTR-dependent activation of the target promoter. The LTTR binds to alternate sites within the ABS in response to effector-induced changes in the conformation of the LTTR. The effector-free and effector-bound conformations of the LTTR, bound to alternate sites in the ABS, have different abilities to productively interact with RNA polymerase and stimulate transcription from the target promoter (20, 24).

In this study, these aspects of the LTTR paradigm were tested using the K. pneumoniae ArgP-gdhAp system.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, fusions, growth conditions, and in vivo assays. All K. pneumoniae strains used in this study were derived from strain KC2653 ({Delta}bla-2 hutC515 str-6), an ampicillin-sensitive (Amps), streptomycin-resistant (Strr) derivative of KC1043 (12) that was obtained from the laboratory of R. A. Bender. Plasmid pCB532 is a derivative of the lacZ fusion vector pRS415 (25) that carries {Phi}(gdhAp'-'lacZ), a transcriptional fusion of the region from –116 to +75, relative to the gdhAp transcription start site, to the lacZ reporter gene (8). Plasmid pCB725 is a derivative of vector pRJ800 (2), which also carries the region from –116 to +75 relative to the gdhAp transcription start site (10).

All derivatives of KC2653 were grown at 30°C in LB medium or W4 minimal medium (12) supplemented with 0.4% (wt/vol) glucose as a carbon source and 0.2% ammonium sulfate plus 0.2% L-glutamine as combined nitrogen sources. Where it is indicated, minimal medium was supplemented with L-lysine HCl, L-arginine HCl, or L-canavanine sulfate to the specified concentrations. Media for plasmid and marker selection were supplemented with antibiotics, as follows: ampicillin (75 µg of the monosodium salt/ml), streptomycin (100 µg of the sulfate salt/ml), kanamycin (50 µg of the sulfate salt/ml), and chloramphenicol (30 µg/ml).

The assay for β-galactosidase was carried out by standard methods as described previously (12) using cultures that were grown to 50 Klett units (approximately 2 x 108 CFU/ml). Specific activities are reported as nanomoles of product formed per minute per milligram of protein (U/mg) and represent the average of three or more assays, with a standard error of the mean (SEM) that is less than 10% of the average.

DNA manipulation. In general, the methods outlined by Maniatis et al. (14) were used to manipulate the DNA. PCR was performed with either Taq DNA polymerase (Gibco-BRL and Invitrogen), as described previously (7), or rTth polymerase (Perkin-Elmer), as specified by the supplier. Custom oligodeoxynucleotide primers were supplied by Gibco-BRL and Invitrogen. Primers for amplifying the previously uncharacterized rbsA, rbsK, argP, and rpiA regions from the K. pneumoniae W70 chromosome were designed using the chromosomal sequence of K. pneumoniae MGH78578 (15), a clinical isolate closely related to K. pneumoniae W70. DNA sequences were determined by using the automated fluorescent dye termination method at the University of Michigan Core facility. DNA fragments carrying substitutions within gdhAp were amplified with mutagenic primers from pCB532 and pCB725, purified, digested with appropriate restriction enzymes, and cloned into pRS415 and pRJ800, respectively. The sequences of all PCR-generated DNA inserts cloned into pRS415 and pRJ800 were confirmed by DNA sequencing.

Construction of a landing pad plasmid for chromosomal insertions at the rbs locus. Plasmid pCB1578 is a so-called "landing pad" vector that facilitates the integration of cloned DNA fragments into the rbs locus on the K. pneumoniae W70 chromosome. This plasmid is a derivative of the pir-dependent allele-exchange vector pKAS46 (26) carrying DNA fragments that were obtained either by PCR or from plasmid pRS415 (Fig. 1A). All derivatives of pKAS46 were replicated in the pir+ E. coli strain BW20767 (16). The construction of pCB1578 involved several intermediate cloning steps employing standard methods. Essentially, 1.4- and 0.9-kb DNA fragments corresponding to the carboxyl terminally truncated rbsA gene (lacking the last 25 codons, {Delta}rbsA75) and the amino terminally truncated rbsK gene (lacking the initiating methionine codon, {Delta}rbsK3), respectively, were amplified from the K. pneumoniae W70 chromosome. These amplified DNA fragments were purified, digested with the appropriate restriction enzymes, and, after intermediate cloning steps, were ultimately cloned into pKAS46 with the same polarity as that found on the K. pneumoniae MGH78578 chromosome. The {Delta}rbsA75 and {Delta}rbsK3 sequences of pCB1578 provide regions of DNA homology to facilitate the recombination of the derivatives of this plasmid into the rbs locus on the K. pneumoniae W70 chromosome. In addition, a 40-bp BamHI-SacI DNA fragment carrying the multiple-cloning region (MCR) from pBC KS(+) (Stratagene) and a 1.2-kb ScaI-BamHI DNA fragment carrying the transcriptional terminators (T1/T2) and the EcoRI restriction site from pRS415 were cloned between {Delta}rbsA75 and {Delta}rbsK3 such that transcription initiated upstream of {Delta}rbsA75 is terminated before the MCR (Fig. 1A). In effect, T1/T2 and MCR carried by pCB1578 replace the rbsB and rbsC genes that lie between rbsA and rbsK on the K. pneumoniae chromosome. Thus, pCB1578 carries a deletion within the rbs operon that lacks the 3' end of rbsA, all of rbsB, all of rbsC, and the 5'end of rbsK, hereafter referred to as {Delta}rbs753. In addition, pCB1578 carries an insertion of T1/T2 and the MCR, hereafter referred to as {Omega}(T1/T2 MCR), between {Delta}rbsA75 and {Delta}rbsK3.


Figure 1
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  		FIG. 1. Plasmids used for allelic replacement on the K. pneumoniae W70 chromosome. pKAS46-based plasmids pCB1578 (A) and pCB1590 (B) carry the pKAS46 backbone (gray arcs) and DNA fragments which were either amplified from the K. pneumoniae W70 chromosome (open arcs with arrowheads) or excised from plasmids (black arcs), where the arrowheads at the ends of arcs indicate the polarity of the open reading frames. The pKAS46 component of these plasmids carries the pir-dependent oriR6K replicon, npt, specifying Kanr; bla, specifying Ampr; and wild-type E. coli rpsL+, conferring Strs. (A) The DNA fragments amplified from the K. pneumoniae chromosome carry the carboxyl terminally truncated rbsA open reading frame ({Delta}rbsA75) and the amino terminally truncated rbsK open reading frame ({Delta}rbsK3). The DNA fragments cloned from pRS415 and pBC KS(+) carry transcriptional terminators (T1/T2, large black arc) and the MCR (MCR) with the indicated restriction sites that are unique on pCB1578 (small black arc), respectively. (B) The DNA fragment amplified from the K. pneumoniae W70 chromosome carries the divergently transcribed rpiA (rpiAKpn) and argP (argPKpn) genes. The argP gene was disrupted by the insertion of a DNA fragment that carries cat, specifying Camr, from pBC KS(+) (black arc) into the argP open reading frame, generating the argP28{Omega}cat allele.

Construction of chromosomal fusions at the rbs locus. All pRS415-based fusions used for in vivo studies were first cloned into the MCR of pCB1578 prior to recombination into the rbs locus on the KC2653 chromosome. Essentially, 3.4-kb DraI-EcoRI DNA fragments carrying either {Phi}(gdhAp'-'lacZ) from pCB532 or comparable pRS415-based plasmids were cloned into pCB1578, which had been digested with SacI, blunt-ended with T4 DNA polymerase, and digested with EcoRI. When the 3.4-kb fragment from pCB532 was cloned into pCB1578, this generated {Delta}rbs753 {Omega}(T1/T2 MCR {Omega}CB532), hereafter referred to as {Delta}rbs753 {Omega}CB532. Derivatives of pCB1578 carrying these fusions were introduced into strain KC2653 by electroporation, as described previously (22), followed by selection for kanamycin resistance (Kanr) and screening for Ampr. These Kanr Ampr isolates carry pCB1578 derivatives that are recombined into the rbs locus of the KC2653 chromosome. The wild-type E. coli rpsL+ gene present on the pKAS46 backbone of these rbs-integrated plasmids confers a dominant Strs phenotype to the KC2653 background strain. Strr derivatives of the Kanr Ampr Strs isolates that have lost the pKAS46 backbone from the rbs-integrated plasmid by homologous recombination were purified after selection on LB plates supplemented with streptomycin and screened for Kan, Amp, and Rbs phenotypes. For this second round of screening, the Rbs phenotype was determined using MacConkey agar (Difco) plates supplemented with 1% (wt/vol) D-ribose. Isolated colonies of purified Strr recombinants that scored as Kans, Amps, and Rbs– were then used for in vivo studies. When {Phi}(gdhAp'-'lacZ) carried by pCB532 was integrated onto the K. pneumoniae chromosome using this procedure, the wild-type rbs allele was replaced by {Delta}rbs375 {Omega}CB532.

Construction of K. pneumoniae argP28. The construction of the K. pneumoniae argP28 allele also utilized the pir-dependent allele-exchange vector pKAS64, with a strategy similar to that used for the integration of lacZ fusions into the rbs locus; however, for this construction, the cat gene, which confers resistance to chloramphenicol (Camr), was inserted into the Eco47-III site within the argP open reading frame. Construction of the pKAS46-based plasmid pCB1590 (Fig. 1B) also involved several intermediate constructs, employing standard methods. Essentially, a 1.9-kb DNA fragment carrying the divergently transcribed rpiA and argP genes was amplified from the K. pneumoniae W70 chromosome, purified, digested with the appropriate restriction enzymes, and, after an intermediate cloning step, was ultimately cloned into pKAS46. The DNA sequence of the cloned argP open reading frame differed from that reported for K. pneumoniae MGH78578 by a single base pair substitution that resulted in a synonymous proline codon change (in strain W70, 5'-CCA-'3; and in strain MGH78578, 5'-CCG-3') at codon position 190 of argP. This single base pair substitution was also found in the cloned DNA sequences of two other argP open reading frames that were independently amplified from the K. pneumoniae W70 chromosome. A 1.4-kb fragment carrying the cat gene was purified from BspHI-digested pBC KS(+), blunt ended, and ligated into the Eco47-III site within argP, generating the argP28{Omega}cat allele, hereafter referred to as argP28. Thus, the DNA fragment carrying the cat gene was inserted after nucleotide 28 of the argP open reading frame in the pKAS46-based pCB1590 plasmid.

Plasmid pCB1590 was introduced into derivatives of KC2653 by electroporation, followed by selection for Kanr and screening for Ampr and Camr. Strr derivatives of the Kanr Ampr Camr isolates were purified after selection on LB plates supplemented with streptomycin and screened for Kan, Amp, and Cam phenotypes. PCR analysis was use to verify the presence of the argP28 allele in recombinants that scored as Strr, Kans, Amps, and Camr. The growth rates of wild-type and argP28 derivatives of KC2653 were similar on all media tested and were not strongly influenced by supplements of either 550 µM lysine, 10 mM arginine, or 500 µM canavanine to the minimal medium. Thus, although wild-type E. coli is modestly sensitive to canavanine, wild-type Salmonella enterica serovar Typhimurium (5) and both the wild-type and the argP28 derivatives of K. pneumoniae KC2653 are relatively canavanine-resistant.

Extract preparation and electrophoretic mobility shift assay. The cells in cultures of K. pneumoniae that were grown to 50 Klett units in minimal medium with the indicated supplements were harvested by centrifugation at 4°C, washed, suspended at 100-fold concentration (about 10 mg of protein/ml) in ice-cold sonication buffer (10 mM imidazole [pH 7.8], 10 mM MgCl2, 10% [vol/vol] glycerol), and subjected to sonic disruption on ice. The cellular debris was pelleted by centrifugation at 10,000 x g at 4°C, and the soluble fraction, hereafter referred to as the extract, was recovered and stored at –20°C. Where the relative amounts of gdhAp-binding activities in extracts were compared, the extracts were prepared on the same day, stored side by side until used, and verified to contain comparable amounts of protein.

For electrophoretic mobility shift experiments, pCB725 and other pRJ800-based derivatives were digested with EcoRI and HindIII, end labeled with [{alpha}-32P]dATP, using the Klenow fragment of DNA polymerase, precipitated with ethanol, and dissolved in distilled water. For these experiments, 4-µl solutions containing 0.1 fmol (0.5 ng) of digested, end-labeled DNA, 1.5 µg double-stranded poly(d[IC]) (Sigma Chemical Co., St. Louis, MO), 75 mM KCl, 5 mM Tris HCl (pH 7.0), 2.5 mM MgCl2, 1 mM dithio-DL-threitol (DTT), 0.5 mM CaCl2, 50 µg acetylated bovine serum albumin (BSA), and where indicated, L-lysine HCl, L-arginine HCl, or L-canavanine sulfate at the specified concentrations were mixed with 1 µl of either sonication buffer or extract. The resulting 5-µl solutions were supplemented with 1 µl of loading solution (15% [wt/vol] Ficoll, 0.25% bromophenol blue, 0.25% xylene cyanol), loaded onto 4% (wt/vol) polyacrylamide-TE (pH 8.4) gels (14), and subjected to electrophoresis at 4°C. After electrophoresis, the gels were dried and subjected to autoradiography.

When the DNA targets carried regions of gdhAp that were less than 50 bp, they were amplified from pCB725 and cloned into pCB1307, a derivative of pRJ800, into which was cloned a 200-bp carrier DNA fragment. The pCB1307 control plasmid and its derivatives carrying the smaller amplified DNA fragments were end-labeled as described above, generating a control target composed of the 200-bp carrier fragment alone and the experimental composite targets composed of the 200-bp carrier fragment ligated to the smaller amplified fragments. These end-labeled DNA preparations were used as targets in mobility shift assays in which the presence of extracts or ArgP did not influence the mobility of the control DNA target (data not shown).

Construction of pCB1603 and the overexpression and purification of ArgP-His. A modified argP open reading frame carrying six CAC (histidine) codons at its 3' end was amplified from the K. pneumoniae W70 chromosome and cloned between the NdeI and EcoRI sites of the thermally inducible pJLA503 expression vector (23), yielding pCB1603. The DNA sequence of the argP open reading frame carried by pCB1603 was identical to that carried by pCB1590, except that it lacked the DNA fragment carrying the cat gene and carried the expected six histidine codons at the 3' of the open reading frame. The modified argP open reading frame of pCB1603 encodes ArgP-His, an affinity-tagged version of the ArgP polypeptide that carries an additional six histidine residues at its carboxyl terminus.

The E. coli strain YMC9 ({lambda}– thi-1 {Delta}lacU169) (1) carrying pCB1603 was grown at 37°C in LB medium supplemented with ampicillin to a density of 25 Klett units, after which the temperature was raised to 42°C to induce the expression of ArgP-His, and incubation was continued to a density of 100 Klett units. The E. coli cells were harvested by centrifugation, washed, suspended at 100-fold concentration in cracking buffer (0.1 M sodium phosphate [pH 7.0], 0.5 M NaCl, 10 mM imidazole, 2.5 mM MgSO4, 10% [vol/vol] glycerol, 1 mM 2-mercaptoethanol) and subjected to sonic disruption on ice. The cellular debris was pelleted by centrifugation at 4°C, and the soluble fraction containing the bulk of the ArgP-His was purified by nickel-affinity chromatography (Amersham and Qiagen) as described previously for the His-tagged NAC protein (22). Fractions (200 µl) were eluted from the affinity column with elution buffer (10% [vol/vol] glycerol, 0.25 M NaCl, 0.25 M imidazole, 0.1 M sodium phosphate, 11 mM 2-mercaptoethanol, 2.5 mM MgSO4 [pH 7.0]) and mixed with an equal volume of glycerol. Fractions containing elevated levels of gdhAp-binding activity were stored at –20°C until used. The storage buffer used to dilute affinity-purified ArgP-His contains equal volumes of elution buffer and glycerol. Purified ArgP-His was more than 90% pure as determined by denaturing polyacrylamide gel electrophoresis (data not shown).

When mobility shift experiments were carried out using affinity-purified ArgP-His, storage buffer was used in place of sonication buffer, and dilutions of purified ArgP-His in storage buffer were used in place of extracts.

DNase I protection assay. The region of gdhAp bound by ArgP-His was determined by using the DNase I protection technique as described previously (8). Essentially, 0.7 µg (0.15 pmol) of end-labeled pCB725 DNA in 32 µl of DNase I digestion buffer (140 mM KCl, 9.3 mM Tris HCl [pH 7.0], 4.7 mM MgCl2, 0.93 mM CaCl2, 47 µg BSA/ml, 1.9 mM DTT, 4.0 µg calf thymus DNA) with or without a 0.7 mM L-lysine HCl supplement was mixed with 8 µl of either storage buffer or diluted samples of affinity-purified ArgP-His. After a 5-min incubation at room temperature, the DNA-protein solutions were mixed with 5 µl of either DNase I dilution buffer or 0.56 mU/µl DNase I (Roche) in DNase I dilution buffer and incubated for 60 s at 37°C. The end-labeled DNA was then precipitated with ethanol, pelleted by centrifugation, dissolved in loading solution (60% [vol/vol] formamide, 4 mM Tris HCl [pH 7.0], 10 mM EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol), heated for 2 min at 90°C, loaded onto a polyacrylamide-urea-Tris-borate-EDTA sequencing gel, and subjected to electrophoresis.


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RESULTS
 
Disruption of the argP gene reduced the activity and the lysine-dependent regulation of gdhAp. The CB532 construct carries {Phi}(gdhAp'-'lacZ), in which the wild-type gdhA promoter is fused to the lacZ reporter gene. The activity of {Phi}(gdhAp'-'lacZ), hereafter referred to as gdhAp, was reduced threefold or more in response to either supplementation of the minimal medium with lysine to 550 µM or disruption of the argP gene (Table 1, KC5998 and KC6540). Although the lysine-dependent regulation of gdhAp was substantially reduced in the absence of a functional argP gene, a limited degree of lysine-dependent regulation persisted in the argP28 KC6540 strain. Additionally, the argP-dependent regulation of gdhAp was essentially abolished in the presence of the lysine supplement. The interrelated natures of the lysine-dependent and the argP-dependent regulations of gdhAp support the suggestion that ArgP is the lysine-sensitive transcriptional activator of gdhAp.


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  		TABLE 1. Specific activities of β-galactosidase directed by gdhAp'-'lacZ fusions as a function of the argP allele and exogenous lysine

The ability of an argP-dependent factor to bind gdhAp is lysine sensitive. Electrophoretic mobility shift assays indicated that the extracts prepared from minimal medium-grown cultures of KC5998 contained an argP-dependent factor that bound the DNA target carrying gdhAp (Fig. 2, lanes 1, 2, and 5). The presence of 550 µM lysine in the growth medium used to prepare the extracts did not strongly influence the quality or quantity of this DNA-binding factor (Fig. 2, lanes 4 and 5). However, the presence of 55 µM lysine in the DNA-binding solution strongly inhibited the ability of this factor to bind the DNA target (Fig. 2, lanes 5 to 8). These results are consistent with the notion that exogenous lysine accumulates within the cell to levels that inhibit the binding of an argP-dependent transcriptional activator to gdhAp in vivo, effectively abolishing the argP-dependent regulation of gdhAp.


Figure 2
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  		FIG. 2. Electrophoretic mobility shift assay for gdhAp-binding activities. End-labeled DNA (0.1 fmol) from EcoRI-HindIII-digested pCB725 carrying gdhAp was incubated with sonication buffer (lane 1), extracts prepared in sonication buffer (lanes 2 to 8), storage buffer (lane 9), or purified ArgP-His in storage buffer (lanes 10 to 12). The extracts were prepared from cultures of either argP28 strain KC6540 (lane 2) or wild-type strain KC5998 (lanes 3 to 8), grown in minimal medium without (–) (lanes 2 and 5 to 8) or with (+) (lanes 3 and 4) a 550 µM L-lysine HCl supplement. Purified ArgP-His (1.8 pmol monomer/µl) was diluted 81-fold (lane 12) or 243-fold (lanes 10 and 11), and 1-µl portions were added to the DNA-binding solutions. For lanes 2 to 12, the DNA-binding solutions contained the indicated concentrations of L-lysine HCl. The samples were loaded onto a polyacrylamide gel, and the free and shifted DNA fragments were separated by electrophoresis. The positions of the end-labeled DNA were visualized by autoradiography of the dried gels. The labeled arrows at the left indicate the extent of migration of the free and bound DNA targets through the gel.

The presence of 50 µM canavanine in the DNA-binding solution strongly inhibited the ability of this argP-dependent factor to bind gdhAp (data not shown). Although the presence of 100 µM arginine in the DNA-binding solution did not influence the ability of this factor to bind gdhAp, the presence of 1 mM arginine strongly inhibited this interaction (data not shown). The presence of 1 mM 2-oxoglutarate, 1 mM L-glutamine, or 1 mM L-glutamate in the DNA-binding solution did not influence the ability of this factor to bind gdhAp (data not shown). The abilities of lysine, arginine, and canavanine to influence the binding of this argP-dependent factor to gdhAp further suggest that this factor is ArgP.

The lysine-sensitive binding of this factor to gdhAp in vitro is strongly reflected in the lysine-dependent regulation of gdhAp in vivo. In contrast, neither the 10 mM arginine nor the 500 µM canavanine supplement reduced the activity of gdhAp by more than 30% in vivo (data not shown). The relative insensitivity of gdhAp to exogenous arginine and canavanine suggests that these supplements do not accumulate intracellularly to levels that substantially inhibit the binding of ArgP to gdhAp in vivo.

To further test the identity of this argP-dependent factor, a modified version of the ArgP polypeptide carrying a histidine affinity tag, ArgP-His, was overexpressed from plasmid pCB1603 and purified by affinity chromatography. Electrophoretic mobility shift assays using purified ArgP-His indicated that such preparations contained high concentrations of an activity whose ability to bind gdhAp was lysine sensitive (Fig. 2, lanes 9 to 12). When elevated levels of ArgP-His were used, an additional bound target band was seen that represents a lower-affinity interaction of ArgP with gdhAp (Fig. 2, lane 12). Additional in vitro studies indicated that this additional interaction involves the region from approximately +20 to +70, relative to the gdhA transcription start site, and is of little physiological significance in vivo (data not shown).

DNase I footprint analysis of ArgP-His at gdhAp. The DNase I footprint analysis of gdhAp indicated that purified ArgP-His protected the region from approximately –100 to –50 from DNase I digestion and induced a region of DNase I hypersensitivity near position –74 (Fig. 3, lanes 2 to 5, and Fig. 4). Additionally, the presence of 500 µM lysine in the DNA-binding solution abolished these regions of protection and hypersensitivity (Fig. 3, lane 6). Within the promoter distal half of this footprint, a match to the LTTR consensus binding motif (5'-T-N11-A-3') (24) can be found, centered at position –89 (Fig. 4). This pattern of protection and hypersensitivity is typical of that reported for other LTTR proteins (24) and suggests that the regions from approximately –100 to –80 and from approximately –70 to –50 correspond to the RBS and ABS, respectively, for ArgP at gdhAp (Fig. 4). The results of this DNase I footprint analysis provide strong support for the notion that ArgP is the argP-dependent factor whose ability to bind the upstream region of gdhAp is lysine sensitive. Thus, this factor will hereafter be referred to as ArgP.


Figure 3
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  		FIG. 3. DNase I footprinting of ArgP bound to the gdhA promoter region. Portions (0.15 pmol) of end-labeled pCB725 DNA were used undigested (lane 1) or digested with DNase I in the absence (lane 2) or presence of either 0.75 pmol (lane 3), 1.5 pmol (lane 4), or 3.0 pmol (lanes 5 and 6) of purified ArgP-His monomer. In lane 6, the DNase I digestion solution contained 700 µM L-lysine HCl prior to the addition of ArgP-His. The same end-labeled DNA was subjected to partial chemical degradation, generating an A+G sequence ladder (lane A+G). The regions of ArgP-dependent DNase I protection and hypersensitivity are indicated by the boxes superimposed on lanes 3 to 5 and a horizontal arrow to the right of the A+G lane, respectively. The numbers at the right of the A+G lane correspond to nucleotides positions relative to the gdhA transcription start site, as indicated in Fig. 4.


Figure 4
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  		FIG. 4. DNA sequence homologies of ArgP-regulated promoters and summary of substitutions and footprinting data for K. pneumoniae gdhAp. The sequences of K. pneumoniae gdhAp (Kpn gdhAp) from –105 to –46, E. coli gdhAp (Eco gdhAp) from –108 to –49, and E. coli argOp (Eco argOp) from –79 to –21, relative to their respective transcription start sites, are aligned to show the regions of DNA sequence homology. The vertical lines between the sequences indicate nucleotides that are identical in all three sequences. The boxed T and A residues within the DNA sequences represent those of the LTTR consensus binding motif indicated below the sequences. For the K. pneumoniae gdhAp sequence, the approximate extents of the ArgP RBS and ABS (long horizontal brackets) and the ArgP-dependent regions of DNase I protection (solid horizontal bars) are indicated above the sequence. The vertical arrow indicates the approximate position of ArgP-induced DNase I hypersensitivity. The positions of the substitutions A(–96)T, T(–65)A, and GTC(–54 to –52)TAT are indicated above the sequence. For the E. coli argOp sequence, one gap was introduced to improve its homology with the gdhAp sequences.

Substitution analysis of the ArgP binding site. Mobility shift experiments indicated that ArgP bound a DNA target carrying the region of gdhAp from –100 to –51 and a target carrying the region of gdhAp from –100 to –60. In contrast, ArgP did not bind a target carrying the limited region of gdhAp from –116 to –71 (data not shown). These results suggest that the interaction of ArgP with its RBS does not firmly anchor ArgP to gdhAp and that the interaction of ArgP with its ABS is required for ArgP to bind gdhAp. To further define regions of the ArgP binding site that influence ArgP binding, DNA fragments carrying substitutions within the ArgP binding site at gdhAp were amplified and used either to construct chromosomal lacZ fusions or as targets in mobility shift assays (Materials and Methods).

The CB1064 construct carries {Phi}(gdhAp A(–96)T'-'lacZ), in which the modified gdhA promoter carrying the A(–96)T substitution, just upstream of the LTTR consensus binding motif centered at –89, is fused to the lacZ reporter gene (Fig. 4). When it was present in argP+ backgrounds, this substitution reduced gdhAp activity more than twofold and largely abolished the lysine-dependent regulation of gdhAp (8). Although the A(–96)T substitution abolished the argP-dependent regulation of gdhAp, it did not significantly alter the argP-independent activity of gdhAp (Table 1, KC6540, KC6881, and KC6882). The reduced binding of ArgP to the gdhAp target carrying the A(–96)T substitution in vitro (Fig. 5, lanes 2 and 4) is consistent with the argP-dependent defects resulting from this substitution in vivo. Thus, the base pair at –96 appears to provide an interaction that is necessary for ArgP binding in vivo.


Figure 5
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  		FIG. 5. Electrophoretic mobility shift assay for the influences of substitutions within the ArgP binding site on ArgP binding. End-labeled DNA from EcoRI-HindIII-digested pCB725 carrying the wild-type (WT) gdhA promoter (lanes 3 to 6 and 11 to 12) or comparable pRJ800 derivatives carrying modified gdhA promoters (lanes 1 to 2, 7 to 10, and 13 to 14) were incubated with sonication buffer (lanes 1, 3, 5, 7, and 9) or KC5998 extract (lanes 2, 4, 6, and 8 and 10 to 14) prepared as described in Fig. 2. For lanes 12 and 14, the DNA-binding solutions were supplemented with 55 µM L-lysine HCl. After the extract was added to the DNA-binding solution, the samples were subjected to electrophoresis as described in the legend to Fig. 2. The substitutions carried by the modified gdhA promoters are shown below the appropriate lanes at the bottom of the figure and above the K. pneumoniae gdhAp sequence in Fig. 4. The labeled arrows indicate the extents of migration of the free and bound target fragments through the gel.

The CB1938 construct carries {Phi}(gdhAp T(–65)A'-'lacZ), in which the modified gdhA promoter carrying the T(–65)A substitution is fused to the lacZ reporter gene. The T(–65)A substitution lies within the ArgP ABS at gdhAp. This substitution essentially abolished the argP-dependent regulation of gdhAp and increased the argP-independent activity of gdhAp by more than 50% (Table 1, KC6540, KC6923, and KC6924). The reduced binding of ArgP to the gdhAp target carrying the T(–65)A substitution in vitro (Fig. 5, lanes 6 and 10) is consistent with the argP-dependent defects resulting from this substitution in vivo. Thus, the base pair at –65 appears to provide an interaction with the ABS that is necessary for ArgP binding in vivo.

The CB1703 construct carries {Phi}(gdhAp GTC(–54 to –52)TAT'-'lacZ), in which the modified gdhA promoter carrying the GTC(–54 to –52)TAT substitution is fused to the lacZ reporter gene. The GTC(–54 to –52)TAT substitution together with the thymidine residue at position –65 of gdhAp generates the LTTR consensus binding motif centered at –59 within the ArgP ABS at gdhAp (Fig. 4). The GTC(–54 to –52)TAT substitution did not strongly alter the argP-independent activity of gdhAp (Table 1, KC6540 and KC6907). Nor did the substitution strongly alter the approximately fourfold lysine-dependent decrease in gdhAp activity in the argP+ background (Table 1, KC5998 and KC6410). However, this substitution did facilitate the argP-dependent regulation of gdhAp in cultures grown in the presence of exogenous lysine (Table 1, KC6410 and KC6907). Additionally, this substitution more than doubled the activities of gdhAp when they were determined in cultures of argP+ strains grown in both the absence and the presence of 550 µM lysine (Table 1, KC5998 and KC6410). The GTC(–54 to –52)TAT substitution did not strongly increase the binding of ArgP to gdhAp in vitro when lysine was not present in the DNA-binding solution (Fig. 5, lanes 6, 8, 11, and 13). However, this substitution did substantially increase the binding of ArgP to gdhAp in vitro when the DNA-binding solution contained 55 µM lysine (Fig. 5, lanes 12 and 14). As the GTC(–54 to –52)TAT substitution appeared to reduce the influences of lysine on ArgP-gdhAp interactions in vivo and in vitro, these results are consistent with the notion that this substitution provides additional interactions that increase the binding of at least the lysine-bound conformation of ArgP to gdhAp in vivo.


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DISCUSSION
 
Role of ArgP in the lysine-dependent regulation of gdhAp. This report identifies ArgP, the product of the argP gene, as the lysine-sensitive factor postulated to bind the upstream region of gdhAp and stimulate promoter activity approximately threefold when K. pneumoniae is grown in minimal medium (8). The parallels between the lysine-sensitive binding of ArgP to gdhAp in vitro and the lysine-dependent regulation of gdhAp in vivo suggest a mechanism where, during growth in minimal medium, exogenous lysine accumulates within the K. pneumoniae cytoplasm. At high intracellular concentrations, lysine binds to and causes ArgP to adopt a conformation that has a reduced affinity for its binding site at gdhAp. Such a lysine-mediated reduction in affinity might effectively prevent ArgP from binding to its site at gdhAp. In the absence of binding to its site at gdhAp, ArgP is unable to interact productively with the transcriptional apparatus and stimulate transcription from gdhAp. Such a mechanism provides a simple yet effective mechanism by which exogenous lysine can limit GDH levels in enteric bacteria.

The lysine-sensitive binding of ArgP to gdhAp represents an interesting variation from the emerging paradigm by which many members of the LTTR family regulate their target promoters (see the introduction), in that ArgP does not appear to be firmly anchored to its RBS at gdhAp by the interactions that typically occur between the LTTR and the RBS. This inability of ArgP to anchor to the RBS at gdhAp makes the binding of ArgP to gdhAp dependent upon the strength of what are normally secondary interactions between the LTTR and the ABS. The data presented here suggest that the effector-free conformation of ArgP is able to interact with the ABS in a manner that compensates for the atypically weak ArgP-RBS interaction. In contrast, the lysine-bound conformation of ArgP appears to be unable to interact with the ABS in a similar manner. Thus, the ability of lysine to inhibit the binding of ArgP to gdhAp in vitro is consistent with an atypically low affinity of ArgP for its RBS and a substantial difference in the affinities of the effector-free and lysine-bound conformations of ArgP for the ABS.

Role of ArgP in the weak NAC-mediated repression of gdhAp. As ArgP appears to be the lysine-sensitive activator of gdhAp, ArgP must also be the target of the weak repression of gdhAp that occurs when NAC is expressed in minimal medium-grown K. pneumoniae (8). The features of this weak NAC-mediated repression suggest that it is facilitated by the mutually exclusive binding of NAC and ArgP to the upstream region of gdhAp (8, 22). The binding site of ArgP at gdhAp, which spans the region from –100 to –50 of gdhAp, overlaps the upstream binding site of NAC at gdhAp, which spans the region from –100 to –75 of gdhAp (8). The overlapping nature of the ArgP and NAC binding sites, as well as the abilities of the A(–96)T substitution to reduce the binding of NAC (8) and the binding of ArgP to gdhAp in vitro, is consistent with the notion that the binding of NAC and ArgP to the upstream region of gdhAp might be mutually exclusive. Thus, NAC and lysine may directly prevent ArgP from binding to its site at gdhAp in vivo.

The atypically weak ArgP-RBS interaction at gdhAp conceivably limits the stability of the ArgP-gdhAp complex in vivo, even during culturing in minimal medium lacking lysine. The limited stability of this complex might increase the frequencies at which ArgP disassociates from its site at gdhAp, and NAC can bind to its site centered at –89. Thus, the atypically weak interaction of ArgP with its RBS at gdhAp might facilitate the ability of NAC and of lysine to directly prevent the binding of ArgP to its site at gdhAp in vivo.

Roles of binding site sequences in the binding of ArgP to DNA. Although several reports have described the binding of ArgP, also designated IciA, to DNA (11), only the report of the binding of ArgP to argOp in E. coli describes the influences of low-molecular-weight effectors on this interaction in vitro (11). Only a few aspects of the binding of ArgP to argOp in E. coli are also seen in the binding of ArgP to gdhAp in K. pneumoniae. Of these aspects, it should be noted that the effector-free conformation of ArgP binds both target promoters in vitro. Of the many differences, perhaps the one aspect that most strongly distinguishes these two ArgP-DNA binding interactions is that lysine increases the affinity of ArgP for its binding site at argOp in E. coli more than threefold (11). This difference suggests that ArgP interacts with its binding sites at these promoters in substantially different manners. The contribution of the 4% divergence of the amino acid sequences of the K. pneumoniae and the E. coli ArgP polypeptides to this difference has not yet been assessed. However, differences between the DNA sequences of the ArgP binding sites may strongly contribute to the different interactions between the ArgP proteins and their binding sites at these promoters.

The E. coli ArgP protein binds the region from approximately –85 to –20, relative to the E. coli argO transcription start site (11). The DNA sequences of the ArgP binding sites at the E. coli argO and the K. pneumoniae gdhA promoters share a common promoter distal sequence, 5'-AT-N3-TTTT-N4-AT-3', centered at positions –63 and –89, respectively (Fig. 4). This common promoter distal sequence is centrally located within the regions corresponding to the ArgP RBS of these promoters and contains the LTTR consensus binding motif that, though often necessary, is itself insufficient for LTTR binding (Fig. 4). Additionally, the first deoxy-adenosine residue of this common RBS-associated sequence corresponds to that at position –96 of the K. pneumoniae gdhA promoter (Fig. 4), which is required for the interaction of ArgP with the K. pneumoniae gdhA promoter in vivo and in vitro. This common RBS-associated sequence may provide interactions that are required for the binding of ArgP to these promoters.

The activity of the E. coli gdhA promoter is stimulated in an argP-dependent, lysine-sensitive manner in vivo (T. Goss, manuscript in preparation). In addition, the E. coli gdhA promoter is bound by an argP-dependent factor whose ability to bind the promoter region is lysine sensitive (T. Goss, manuscript in preparation). The RBS-associated sequence that is common to both the K. pneumoniae gdhA and the E. coli argO promoters is also found in the upstream region of the E. coli gdhA promoter (http://www.ncbi.nlm.nih.gov/GenBank/index.html) (Fig. 4). Although the DNA sequence of the region from –100 to –80 of the K. pneumoniae gdhA promoter is more than 80% identical to the comparable region of the E. coli gdhA promoter, these regions are less than 50% identical to the comparable region of the E. coli argO promoter (Fig. 4). Despite the presence of the common RBS-associated sequence, the divergence of this region of argOp from the comparable regions of the two gdhA promoters suggests that the DNA sequence of the ArgP RBS may contribute to the different abilities of these regions to strongly anchor ArgP to the argO and gdhA promoters in vivo during growth in the presence of exogenous lysine.

When a single nucleotide gap is introduced in the sequence of the E. coli argO promoter to maximize its homology to the sequences of the two gdhA promoters, all three promoters contain the common promoter proximal sequence 5'-TTATNAATTT-3' (Fig. 4). This common promoter proximal sequence is asymmetrically positioned within the regions corresponding to the ArgP ABS of the K. pneumoniae gdhA and E. coli argO promoters (Fig. 4). The third thymidine residue of this common ABS-associated sequence corresponds to that at position –65 of the K. pneumoniae gdhA promoter (Fig. 4) that is required for the interaction of ArgP with the K. pneumoniae gdhA promoter in vivo and in vitro. This common ABS-associated sequence may be important for the ArgP-DNA interactions that are common to the gdhA and argO promoters, suggesting that this sequence facilitates binding of ArgP to all three of these promoters in vivo, during growth in the absence of exogenous lysine.

The DNA sequences of all three promoters diverge substantially in the regions comparable to the span from –58 to –50 of the K. pneumoniae gdhAp promoter (Fig. 4). Within these divergent regions, neither gdhAp contains deoxy-adenosine residues at positions comparable to that at position –28 of argOp. Thus, neither gdhAp contains LTTR consensus binding motifs at positions comparable to that centered at –34 at argOp (Fig. 4). The GTC(–54 to –52)TAT substitution at the K. pneumoniae gdhA promoter introduces a deoxy-adenosine residue at a position comparable to that at position –28 of argOp and generates the LTTR consensus binding motif at a position comparable to that centered at –34 at argOp (Fig. 4). Thus, the GTC(–54 to –52)TAT substitution improves the homology of the region from –58 to –50 of the K. pneumoniae gdhA promoter to the comparable region of argOp. The GTC(–54 to –52)TAT substitution also improves the interaction of ArgP with gdhAp in the presence of lysine, in vivo and in vitro. The extent of DNA sequence homology between these regions of the E. coli argO and the K. pneumoniae gdhA promoters parallels the abilities of these promoters to interact with ArgP in the presence of lysine. This parallel suggests that the DNA sequence of the promoter proximal half of the ArgP ABS at the gdhA and argO promoters contributes to the different affinities of ArgP for these promoters in vivo, during growth in the presence of exogenous lysine.

Finally, it should be noted that although the effector-free conformations of ArgP bind gdhAp and argOp in vitro, this conformation of ArgP appears to stimulate gdhAp, yet fails to stimulate argOp (11). As the ArgP binding site at gdhAp is approximately 30 bp farther upstream from the transcription start site than that at argOp, this architectural difference may influence the ability of effector-free ArgP to stimulate transcription in vivo.

Role of lysine in the regulation of gdhAp. The lysine-dependent regulation of gdhAp influences the cell's capacity to make glutamate, which serves roles as counterion to K+ in osmotic pressure homeostasis (reviewed in reference 6) and primary amino group donor in the biosynthesis of approximately 80% of all nitrogenous compounds in the cell (reviewed in reference 21). As the intracellular levels of glutamate required for osmotic homeostasis might differ from those required for biosynthesis under various growth conditions, the extent to which glutamate fulfills these two roles may be independently monitored by means that do not reflect glutamate levels per se. The extent to which glutamate fulfills its requirement for the biosynthesis of other nitrogenous compounds might be signaled by the intracellular levels of one or more key nitrogenous compounds. Of the 20 common amino acids, lysine is noteworthy in that, despite its relatively high nitrogen content, enteric bacteria do not readily use lysine as a source of nitrogen or even carbon (4, 9, 28). Despite its lack of utility as a growth substrate, lysine can be decarboxylated to cadaverine, which is either used as a polyamine (5) or excreted as a means of neutralizing an acidic environment (27). Additionally, the size of the intracellular pool of lysine is likely to be related to that of its immediate precursor, diaminopimelate, which is required for cell wall biosynthesis (19). These unique roles and features of lysine could account for its possible use as one such key nitrogenous compound that may serve as a surrogate for glutamate in signaling the extent of glutamate overflow from osmotic pressure homeostasis into the biosynthetic pathways for other nitrogenous compounds. In such a capacity, lysine levels may be one of several signals that mediate the feedback regulation of gdhAp, allowing enteric bacteria to fine tune their glutamate biosynthetic capacities to achieve both osmotic and biosynthetic homeostasis.

NAC and ArgP are the only factors currently known to directly bind and regulate gdhAp in enteric bacteria. The regulation of gdhAp by NAC and ArgP provides a rare example of the physiologically relevant, opposing regulation of a promoter by two dissimilar LTTR family members.


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ACKNOWLEDGMENTS
 
I thank Robert A. Bender for motivation, funding, and useful discussions. I also thank Anthony Schwacha for making KC2653 and Brian K. Janes and Daniel Heikka for the initial steps of the procedure that yielded pCB1578.

This work was supported by Public Health Service grant GM47156 from the National Institutes of Health to R. A. Bender.


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FOOTNOTES
 
* Mailing address: Department of Molecular, Cellular and Developmental Biology, the University of Michigan, Ann Arbor, MI 48109-1048. Phone: (734) 763-4681. Fax: (734) 647-0884. E-mail: taaj{at}umich.edu Back

{triangledown} Published ahead of print on 18 April 2008. Back


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REFERENCES
 
    1
  1. Backman, K., Y.-M. Chen, and B. Magasanik. 1981. Physical and genetic characterization of the glnA-glnG region of the Escherichia coli chromosome. Proc. Natl. Acad. Sci. USA 78:3743-3747.[Abstract/Free Full Text]
    2. 2
  2. Ball, C. A., R. Osuna, K. C. Ferguson, and R. C. Johnson. 1992. Dramatic changes in Fis levels upon nutrient upshift in Escherichia coli. J. Bacteriol. 174:8043-8056.[Abstract/Free Full Text]
    3. 3
  3. Bender, R. A., P. M. Snyder, R. Bueno, M. Quinto, and B. Magasanik. 1983. Nitrogen regulation system of Klebsiella aerogenes: the nac gene. J. Bacteriol. 156:444-446.[Abstract/Free Full Text]
    4. 4
  4. Brenchley, J. E., M. J. Prival, and B. Magasanik. 1973. Regulation of the synthesis of enzymes responsible for glutamate formation in Klebsiella aerogenes. J. Biol. Chem. 248:6122-6128.[Abstract/Free Full Text]
    5. 5
  5. Charlier D., and N. Glansdorff. September 2004, posting date. Chapter 3.6.1.10, Biosynthesis of arginine and polyamines. In R. Curtiss III et al. (ed.), EcoSal-Escherichia coli and Salmonella: cellular and molecular biology. ASM Press, Washington, DC. http://www.ecosal.org.
    6. 6
  6. Csonka, L. N., and W. Epstein. 1996. Osmoregulation, p. 1210-1217. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology Press, Washington, DC.
    7. 7
  7. Goss, T. J., A. Perez-Matos, and R. A. Bender. 2001. Roles of glutamate synthase, gltBD, and gltF in nitrogen metabolism of Escherichia coli and Klebsiella aerogenes. J. Bacteriol. 183:6607-6619.[Abstract/Free Full Text]
    8. 8
  8. Goss, T. J., B. K. Janes, and R. A. Bender. 2002. Repression of glutamate dehydrogenase formation in Klebsiella aerogenes requires two binding sites for the nitrogen assimilation control protein, NAC. J. Bacteriol. 184:6966-6975.[Abstract/Free Full Text]
    9. 9
  9. Gutnick, D., J. M. Calvo, T. Klopotowski, and B. N. Ames. 1969. Compounds which serve as the sole source of carbon or nitrogen for Salmonella typhimurium LT-2. J. Bacteriol. 100:215-219.[Abstract/Free Full Text]
    10. 10
  10. Janes, B. K., P. J. Pomposiello, A. Perez-Matos, D. J. Najarian, T. J. Goss, and R. A. Bender. 2001. Growth inhibition caused by overexpression of the structural gene for glutamate dehydrogenase (gdhA) from Klebsiella aerogenes. J. Bacteriol. 183:2709-2714.[Abstract/Free Full Text]
    11. 11
  11. Laishram, R. S., and J. Gowrishankar. 2007. Environmental regulation operating at the promoter clearance step of bacterial transcription. Genes Dev. 21:1258-1272.[Abstract/Free Full Text]
    12. 12
  12. Macaluso, A., E. A. Best, and R. A. Bender. 1990. Role of the nac gene product in the nitrogen regulation of some NTR-regulated operons of Klebsiella aerogenes. J. Bacteriol. 172:7249-7255.[Abstract/Free Full Text]
    13. 13
  13. Magasanik, B. 1993. The regulation of nitrogen utilization in enteric bacteria. J. Cell Biochem. 51:34-40.[CrossRef][Medline]
    14. 14
  14. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
    15. 15
  15. McClelland, M., E. K. Sanderson, J. Spieth, W. S. Clifton, P. Latreille, A. Sabo, K. Pepin, V. Bhonagiri, S. Porwollik, J. Ali, and R. K. Wilson. September 2006, posting date. Klebsiella pneumoniae subsp. pneumoniae MGH 78578 project at the Klebsiella pneumonia Genome Sequencing Project. Genome project ID:31. http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genomeprj&cmd=showDetail=View&TermToSearch=31 .
    16. 16
  16. Metcalf, W. W., W. Jiang, L. L. Daniels, S.-K. Kim, A. Haldimann, and B. L. Wanner. 1996. Conditionally replicative and conjugative plasmids carrying lacZ for cloning, mutagenesis, and allele replacement in bacterial. Plasmid 35:1-13.[CrossRef][Medline]
    17. 17
  17. Nandineni, M. R., and J. Gowrishankar. 2004. Evidence for an arginine exporter encoded by yggA (argO) that is regulated by the LysR-type transcriptional regulator ArgP in Escherichia coli. J. Bacteriol. 186:3539-3546.[Abstract/Free Full Text]
    18. 18
  18. Nandineni, M. R., R. S. Laishram, and J. Gowrishankar. 2004. Osmosensitivity associated with insertions in argP (iciA) or glnE in glutamate synthase-deficient mutants of Escherichia coli. J. Bacteriol. 186:6391-6399.[Abstract/Free Full Text]
    19. 19
  19. Park, J. T. 1996. The murine sacculus, p. 48-57. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology Press, Washington, DC.
    20. 20
  20. Porrúa, O., M. Garcia-Jaramillo, E. Santero, and F. Govantes. 2007. The LysR-type regulator AtzR binding site: DNA sequences involved in activation, repression and cyanuric acid-dependent repositioning. Mol. Microbiol. 66:410-427.[CrossRef][Medline]
    21. 21
  21. Reitzer, L. July 2004, posting date. Chapter 3.6.1.3, Biosynthesis of glutamate, aspartate, asparagine, L-alanine, and D-alanine. In R. Curtiss III et al. (ed.), EcoSal–Escherichia coli and Salmonella: cellular and molecular biology. ASM Press, Washington, DC. http://www.ecosal.org.
    22. 22
  22. Rosario, C. J., and R. A. Bender. 2005. Importance of tetramer formation by the nitrogen assimilation control protein for strong repression of glutamate dehydrogenase formation in Klebsiella pneumoniae. J. Bacteriol. 187:8291-8299.[Abstract/Free Full Text]
    23. 23
  23. Schauder, B., H. Blocker, R. Frank, and J. E. G. McCarthy. 1987. Inducible expression vectors incorporating the Escherichia coli atpE translation initiation region. Gene 29:279-283.[Medline]
    24. 24
  24. Schell, M. A. 1993. Molecular biology of the LysR family of transcriptional regulators. Annu. Rev. Microbiol. 47:597-626.[CrossRef][Medline]
    25. 25
  25. Simons, R. W., F. Houman, and N. Kleckner. 1987. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53:85-96.[CrossRef][Medline]
    26. 26
  26. Skorupski, K., and R. K. Taylor. 1996. Positive selection vectors for allelic exchange. Gene 169:47-52.[CrossRef][Medline]
    27. 27
  27. Slonczewski, J. L., and J. W. Foster. 1996. pH-regulated genes and survival at extreme pH, p. 1539-1549. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology Press, Washington, DC.
    28. 28
  28. Tyler, B. 1978. Regulation of the assimilation of nitrogen compounds. Annu. Rev. Biochem. 47:1127-1162.[CrossRef][Medline]

Journal of Bacteriology, June 2008, p. 4351-4359, Vol. 190, No. 12
0021-9193/08/$08.00+0     doi:10.1128/JB.00295-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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