JBC Invitrogen Ultrasensitive Cytokine Assays

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Originally published In Press as doi:10.1074/jbc.M404348200 on May 24, 2004

J. Biol. Chem., Vol. 279, Issue 32, 33253-33262, August 6, 2004
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Regulation of Penicillin G Acylase Gene Expression in Escherichia coli by Repressor PaaX and the cAMP-cAMP Receptor Protein Complex*

Hyoung Seok Kim{ddagger}, Tae Sun Kang§, Joon Sik Hyun§, and Hyen Sam Kang

From the Department of Microbiology, School of Biological Sciences, Seoul National University, San 56-1, Shillim-dong, Kwanak-gu, 151-742, Korea

Received for publication, April 20, 2004 , and in revised form, May 14, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The pga gene of Escherichia coli W ATCC11105 encodes a penicillin G acylase whose expression is regulated at both the transcriptional and post-transcriptional level. In this work we have shown that PaaX is the repressor of pga expression, and we have identified its binding consensus as TGATTC(N27)GAATCA. We conclude that the process of "PAA induction" actually involves relief of pga from repression by PaaX. Other features of the pga promoter have also been characterized. (i) It has a native class III cAMP-receptor protein (CRP)-dependent promoter with two CRP-binding sites. (ii) The downstream CRP-binding site II has higher affinity. (iii) Binding of cAMP-CRP to both sites (I + II) is required for maximal expression. We have also shown that the PaaX-binding site overlaps with the CRP-binding site I. This implies that PaaX and the cAMP-CRP compete for binding to the region around the CRP-binding site I and therefore have antagonistic effects on pga expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Penicillin G acylase (PGA,1 EC 3.5.1.11 [EC] ) is a type II penicillin acylase that hydrolyzes penicillin G to 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA) (1). It is one of the most important industrial enzymes for the production of semi-synthetic penicillins (2). PGAs have been found in numerous bacteria and fungi (3-8), and the PGA of Escherichia coli W ATCC11105 is the best characterized (2, 9). In free-living E. coli, PGA is thought to act as a scavenger enzyme for many different natural esters and amides of PAA and its derivatives, such as hydroxyphenylacetic acid (2, 10). Thus, when E. coli encounters phenylacetylated compounds, the periplasmic PGA degrades them to PAA, and this moiety then diffuses into the nucleoplasm. PaaK, the PAA-CoA ligase, converts the PAA to PAA-CoA (11), which in turn inhibits PaaX, the repressor of the paa operon, from binding to DNA (12). As a consequence the paa-operon is expressed, and the meta-cleavage pathway subsequently degrades PAA-CoA before it enters the tricarboxylic acid cycle (13).

The expression of E. coli pga is regulated by many factors such as temperature (14, 15), oxygen (16-18), carbon source (19-21), and PAA (22). It is also controlled at the level of translocation (18, 23, 24), auto-processing (25-27), and stabilization by chaperons (28-30). E. coli PGA is the first example of auto-proteolytic processing of an inactive precursor to a functional protein in bacteria (31), and this occurs in a pH- and temperature-dependent manner (15, 32). According to the x-ray crystallographic analysis (26), PGA belongs to the family of N-terminal nucleophile hydrolases (33).

Previous studies of the E. coli pga promoter identified two putative CRP-binding sites (14, 34) and two possible IHF-binding sites (21); however, there is no direct evidence that CRP or IHF proteins bind to the promoter (35). It has been inferred that the pga promoter is a class III CRP-dependent promoter (21).

In the present work, we have clarified several important features of the regulation of pga expression. First, we have shown that PaaX is a transcriptional repressor of pga expression. Second, we have identified the PaaX-binding consensus sequence. Third, these findings have led us to characterize "PAA induction," the increase of PGA production in the presence of PAA, as primarily resulting from relief of pga from repression by PaaX. Fourth, we have demonstrated that the pga promoter is a class III CRP-dependent promoter. Finally, we have shown that the PaaX repressor and CRP activator compete for binding to the pga promoter in a region around the upstream CRP-binding site (site I), and we have established that this competition for binding underlies the mechanism of transcriptional regulation of pga in response to PAA.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains, Plasmids, Primers, and Chemicals—The bacterial strains, plasmids, and primers used in this study are listed in the Supplemental Material. pUCK was constructed by inserting the kanamycin resistance gene into the {beta}-lactamase gene of pUC18 (36). All primers were synthesized by GenoTech. Chemicals for media were purchased from Difco, and we bought other chemicals from Sigma unless stated.

Growth Media and Culture Condition—E. coli W ATCC11105 and its derivatives were grown at 30 °C with shaking at 180 rpm in M9 minimal medium (37) supplemented with 0.2% glycerol or 0.2% glucose as sole carbon source; when required, 0.05% PAA was added. Other standard molecular biological procedures were performed according to Sambrook and Russel (38) by using E. coli DH5{alpha} cells grown at 37 °C with shaking at 200 rpm in LB medium containing 0.5 mM isopropyl-1-thio-{beta}-D-galactopyranoside and 40 µg/ml 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside (X-gal). When required, media were solidified with 2% agar powder. Antibiotics were added at the following concentrations as appropriate: 50 µg/ml ampicillin, 34 µg/ml chloramphenicol, 50 µg/ml kanamycin, and 10 µg/ml tetracycline.

Construction of Plasmids—Various plasmids harboring the pga promoter derivatives were constructed in two steps. First, the largest DNA fragment of pUCK-C1 was generated by PCR (AccuPower PCR PreMix, Bioneer) with the genomic DNA of E. coli W ATCC11105 as template and the up-700-EcoRI/down-1-PstI primer set. The PCR product was column-purified (Nucleogen), digested with EcoRI (EcoRI restriction sites were introduced into all up-series primers) and PstI (PstI restriction sites were introduced into all down-series primers), re-purified through a column, and inserted into pUCK digested with the same restriction enzymes and column-purified. After transformation, colonies harboring the recombinant plasmid were identified and confirmed by sequencing. Second, the smaller DNA fragments were also obtained by PCR using pUCK-C1 as template with the relevant primer sets and subsequent digestion with DpnI (New England Biolabs) to degrade the template. The PCR products were treated as in the case of pUCK-C1, and the corresponding plasmids were constructed.

To overexpress the recombinant paaX and crp genes, it was cloned from the genomic DNA of E. coli W by PCR and inserted into pET-30a(+) (Novagen) so that it was tagged with the His6 tag and several linker amino acids. PCR was carried out with the paaX-NcoI-5/paaX-EcoRI-3 and crp-NcoI-5/crp-EcoRI-3 primer sets, and the product was column-purified, digested with NcoI and EcoRI, re-purified, and inserted into pET-30a(+) digested with the same restriction enzymes and column-purified. Similarly, pUC-paaK and pUC-paaX that overexpress recombinant paaK and paaX were made by PCR with paaK-EcoRI-5/paaK-EcoRI-3 and paaX-EcoRI-5/paaX-EcoRI-3 primer sets and subsequent subcloning into pUC18.

pMAK705 derivatives were constructed in three steps. First, the pga promoter in pUCK-C3 had undergone site-directed mutagenesis. Second, PCR was carried out with pUCK-C3 derivatives as templates and up-200-EcoRI/down-1-EcoRI primer set. The PCR products were column-purified, digested with EcoRI, re-purified, and inserted into pRS415 (39) digested with the same restriction enzyme and column-purified. The orientation of promoters was confirmed by PCR with up-200-EcoRI/lacZ-down-BamHI primer set. Third, by using the up-200-BamHI/lacZ-down-BamHI primer set, Ppga::lacZ fragments were obtained and digested with BamHI that was subsequently inserted into pMAK705 (40) digested with the same restriction enzyme.

pMAK-705-del-lacZ plasmid was constructed in three steps. First, the upstream and downstream regions of the lacZ open reading frame were obtained by PCR with the genomic DNA of E. coli W as template and lacZ-del-up-5/lacZ-del-up-3 and lacZ-del-down-5/lacZ-del-down-3 primer sets, respectively. Second, they were digested with AscI and ligated with the equal amount of each fragments. Third, ligated fragments were digested with BamHI and inserted into pMAK705 digested with the same restriction enzyme. Several colonies were analyzed by PCR with lacZ-del-up-5/lacZ-del-down-3 primer set to confirm the existence of each upstream and downstream region. For ChIP assays, we constructed pREP42-crp and pREP42-paaX plasmids with crp-BglII-5/crp-NotI-3 and paaX-BglII/paaX-NotI-3 primer sets, respectively, in a similar way as above.

Site-directed Mutagenesis—PCR-based, site-directed mutagenesis was performed with a QuikChange site-directed mutagenesis kit (Stratagene). The primers that already contain an altered base at the desired site were synthesized according to the manufacturer's instructions, but their size was adjusted to enhance the specificity of annealing during PCR. PCR was performed with pUCK-C3 as template and the relevant primer sets, and PCR products were digested with DpnI to degrade the template. Colonies harboring the mutagenized plasmid were identified by mini-prep and sequencing. When another mutation has to be introduced, PCR was performed with single base substituted plasmid as template.

Construction of Mutant Cell Lines—We constructed deletion mutants of E. coli W ATCC11105 by homologous recombination between a PCR-generated linear DNA fragment containing an antibiotic resistance gene and chromosomal DNA, catalyzed by the {lambda} RED system (41). E. coli W ATCC11105 cells were transformed with pIJ790 (42), and electrocompetent cells were prepared as described (41) with some modifications. (i) The cells were cultured in LB medium. (ii) L-Arabinose was used to induce the {lambda} RED system at a concentration of 100 mM. The linear DNA fragments were obtained by PCR with 70-base primers that consisted of the following two regions: a 5' 50-base region complementary to part of the target gene, and a 3' 20-base region complementary to the tetracycline resistance gene. PCR was performed with pBR322 plasmid as template, and the product was completely digested with DpnI and column-purified. Electroporation was carried out with a Gene-Pulser (Bio-Rad) with a 0.1-cm cuvette containing 50 µl of electrocompetent cells and 5 µl of PCR product. After shocking at 1.8 kV, 1 ml of ice-cold LB medium was added immediately, and the cells were incubated at 37 °C with shaking at 200 rpm for 1 h. The regenerated cells were spread on an LB agar plate containing tetracycline and incubated at 37 °C for 24 h. Colonies were streaked onto a new plate for further purification, and several clones were analyzed by PCR to confirm deletion of the target gene. Confirmed deletion strains were cultured in 50 ml of LB medium at 42 °C with shaking at 200 rpm for 24 h to prevent the multiplication of pIJ790 that depends on a temperature-sensitive replication origin. 0.5 ml of the cultured cells were transferred to fresh medium and further incubated under the same conditions. After three successive cultures, the cells were diluted and plated to isolate single colonies. Single colonies were tested for sensitivity to tetracycline to confirm the absence of pIJ790.

Penicillin G Acylase Assays—The penicillin G acylase assays were performed as described previously (32, 43).

{beta}-Galactosidase Assays—{beta}-Galactosidase activities of E. coli W, grown at 30 °C on a shaking incubator, were measured as described (37).

RT-PCR Analysis—Total RNA from E. coli W ATCC11105 cells was prepared with an RNeasy mini kit (Qiagen). The appropriate conditions were determined empirically at various points during growth. The quality of the total RNA was checked both by formamide gel electrophoresis visualized by EtBr staining and PCR with the up-200-EcoRI/down-1-PstI primer set, which is complementary to the nontranscribed region of the pga gene. When required, 10 units of RQ1 RNase-free DNase (Promega) was added to 10 µg of total RNA and incubated at 37 °C for at least 4 h to degrade contaminating genomic DNA. After clean-up using the same kit, RNA purity was reconfirmed. The DNA-free total RNA was then used in RT-PCR with a one-step RT-PCR kit (Qiagen). Reaction mixtures contained 1 ng of total RNA and 30 pmol each of the pga-RNA-5/pga-RNA-3 primer set, which is complementary to the 3' 800-base region of the pga structural gene. 30-(Fig. 5) or 35-cycle (Fig. 1) reactions were carried out.



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  		FIG. 5.
PAA relieves PaaX repression of pga expression. The PGA activities of E. coli W wild type (WT) (panel A) and its two mutants (panels B and C) were measured during growth in M9 minimal medium containing glycerol. All values are averages of three independent measurements, and standard deviations did not exceed 25%. Symbols are as follows: squares, cells grown without PAA; triangles, cells grown with PAA; filled symbols, growth (A600 value); and open symbols, PGA activity. RT-PCR analysis (panel D) was performed with total RNA prepared from wild type cells in panel A. Lane 1, cells grown without PAA, sampled at A600 {approx} 0.2; lanes 2-4, cells grown with PAA, sampled at A600 {approx} 0.2, 0.8, and 2.0, respectively. SM, size marker.

 



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  		FIG. 1.
Evidence that PaaX is the transcriptional repressor of pga expression. Panel A, PGA activity of wild type (WT), {Delta}paaX, {Delta}paaK, and {Delta}paaX cells complemented by pUC-paaX. All values are averages of three independent experiments. Standard deviations, shown by bars, did not exceed 20%. Panel B, RT-PCR analysis using total RNA extracted from wild type, {Delta}paaX, and {Delta}paaK cells at A600 {approx} 0.2. Three independent experiments gave similar results, and a typical one is shown.

 
Overexpression and Purification of the Recombinant paaX and crp—We transformed E. coli BL21(DE3)pLysS cells with pET-30a(+)-paaX, and a purified transformant was cultured in 3 ml of LB medium supplemented with kanamycin and chloramphenicol at 37 °C with shaking at 200 rpm overnight. The cells were diluted 100-fold in 20 ml of the same medium, grown to A600 of 1, diluted again 100-fold into 2 liters of the same medium, and incubated at 160 rpm. At mid-log phase (A600 = 0.5), 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside was added, and the cells were further incubated for 4 h. They were harvested and washed with TEN buffer (50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA) and lysed with a BugBuster protein extraction reagent plus a Benzonase nuclease (Novagen). His-PaaX was purified with a His·Bind column (Novagen) according to the manufacturer's instructions. The eluates were dialyzed to remove imidazole and adjust the salt concentration. The imidazole concentration could be reduced to 0.5 M without loss of protein, and the same volume of 100% glycerol was then added. Protein samples were dispensed into aliquots and stored at -70 °C. Protein was measured with a Bio-Rad Protein Assay kit, and protein samples were analyzed on 12% SDS-polyacrylamide gels at each step of purification.

In the case of CRP, we transformed E. coli BL21(DE3)pLysS cell with pET-30a(+)-crp and followed the same procedures as PaaX with minor modifications. The final eluates of His-CRP were dialyzed to remove imidazole completely. Subsequently, His-CRP recombinant proteins were cleaved by enterokinase and purified to remove both the His6 tag and enterokinase with an Enterokinase Cleavage Capture kit (Novagen). An intact form of the purified CRP was dispensed into aliquots by adding an equal volume of 100% glycerol and stored at -70 °C.

Electrophoretic Mobility Shift Assay (EMSA)—The various pga promoter derivatives used in EMSAs were prepared by digesting the corresponding plasmids with EcoRI and PstI and purifying them on a Nucleogen column. The fragment from pUCK-C3 (from -200 to -1) as well as others was labeled with [{alpha}-32P]dATP (Amersham Biosciences) with a Prime-a-Gene labeling system (Promega) and purified with a QIAquick nucleotide removal kit (Qiagen). Basic reaction mixtures contained 2 fmol of DNA probe, 0.5 µg of poly(dI-dC), 20 mM Tris-HCl (pH 7.9), 10% glycerol, 100 mM NaCl, 100 mM imidazole, 2 pmol of His-PaaX in a final volume of 40 µl. When required, 200 or 300 fmol of unlabeled promoter construct was added, and PAA or PAA-CoA was provided at the stated concentrations. After incubation for 20 min at 30 °C, reaction mixtures were loaded onto 5% polyacrylamide gels containing 5% glycerol, which had been pre-electrophoresed at 7 V/cm for 30 min, and run at 15 V/cm for 2 h 30 min. The gels were dried onto Whatman 3MM paper and visualized by autoradiography.

In EMSA with CRP proteins, basic reaction mixtures contained 2 fmol of DNA probe, 0.5 µg of poly(dI-dC), 40 mM Tris-HCl (pH 7.9), 1 mM EDTA, 1 mM dithiothreitol, 50 µg/ml bovine serum albumin, 100 mM NaCl, 6 mM MgCl2, 10% glycerol, 4 nmol of cAMP, and CRP proteins in a final volume of 40 µl. After incubation for 20 min at 30 °C, reaction mixtures were loaded onto 5% polyacrylamide gels containing 5% glycerol and 0.1 mM cAMP, which had been pre-electrophoresed at 7 V/cm for 30 min with 0.5x TBE containing 0.1 mM cAMP, and run at 15 V/cm for 3 (Fig. 6, panel A) or for 1 h (Fig. 6, panel B). The gels were dried onto Whatman 3MM paper and visualized by autoradiography.



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  		FIG. 6.
Characterization of CRP-binding site I and II in the pga promoter. EMSAs were performed with pga promoters harboring point mutations (panel A) or deletions (panel B). Lanes 1-4 and lane -cAMP contained 0, 2, 4, 6, and 6 pmol CRP protein, respectively. P and F represent probe used and free probe, respectively; C1 and C2 are the cAMP-CRP binding complexes. The probes used are given in panel C.

 
DNase I Footprinting Experiment—The pga promoter fragment used in DNase I footprinting experiments was prepared from pUCK-C3 as described above. The reaction mixture contained 50 fmol of DNA probe, 1 µg of poly(dI-dC), 20 mM Tris-HCl (pH 7.9), 10% glycerol, 100 mM NaCl, 100 mM imidazole in a final volume of 200 µl. His-PaaX was used at 0, 2, 5, and 10 pmol. After incubation for 20 min at 30 °C, 5 µl of DNase I (Novagen) solution, diluted 10-fold in dilution buffer (50 mM Tris-HCl (pH 7.5), 200 mM MgCl2, 200 mM CaCl2, and 50% glycerol), was added, and incubation was continued for 10 min. To terminate the reaction, 700 µl of DNase I stop solution (5 µl of E. coli tRNA (1 mg/ml) and 50 µl of saturated ammonium acetate in 100% ethanol) were added and rapidly mixed by vortexing vigorously. The reaction mixtures were placed in a dry ice/ethanol bath for 20 min, centrifuged at 4 °C for 20 min to precipitate the DNA, washed twice with ice-cold 70% ethanol, and dried in a speedvac evaporator. The pellets were resuspended in 5 µl of formamide loading buffer (80% formamide, 10 mM NaOH, 1 mM EDTA (pH 8.0), 0.1% xylene cyanol, 0.1% bromphenol blue) and heated at 95 °C for 3 min, followed by immediate quenching on wet ice. Samples were loaded onto 8 or 6% polyacrylamide gels containing 8 M urea for Fig. 3, panels A or B, respectively, pre-electrophoresed at 40 V/cm for 30 min, and run 40 V/cm for 2 h 30 min. Gels were dried onto Whatman 3MM paper and visualized by autoradiography.



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  		FIG. 3.
DNase I footprinting experiment. The probe was the noncoding strand of the pga promoter from -200 to +30, in which His-PaaX protected a single upstream region from digestion by DNase I (panel A). Closer examination showed that the protected region extended from -126 to -77; this region is bracketed as Opga, the pga operator (panel B). Arrow indicates the hypersensitive site in the center of the protected region.

 
Chromatin Immunoprecipitation (ChIP) Assays—The chromatin immunoprecipitation assays for detecting in vivo protein-DNA interactions were done essentially as described (44) with minor modifications. pREP42-paaX and pREP42-crp were transformed with the {Delta}paaX and {Delta}crp, carrying pMAK705 derivatives, respectively, and they were grown to late log phase followed by treatment with 1% formaldehyde and immunoprecipitation with monoclonal mouse anti-c-Myc antibody (Santa Cruz Biotechnology). DNA fragments co-immunoprecipitated with the 2x cMyc-tagged proteins were used as template in PCR to amplify the pga promoter region from -200 to -1, and DNA samples treated without antibody were used as negative control. PCR program with 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s was applied. PCR products were fractionated by 2% agarose gel electrophoresis, stained with ethidium bromide, and photographed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PaaX Is the Repressor of pga Expression—Although E. coli pga has been known for a long time to be negatively regulated (45), the identity of the regulator has not been established. When we transformed E. coli W (PGA+) with the multicopy plasmid pUCK-C, a derivative of pUC18 harboring the upstream pga promoter region from -700 to -22, the constitutive PGA activity of the transformants increased by a factor of 3 (36). This indicated that some repressor was binding to the pga promoter in pUCK-C, causing the concentration of free repressor to fall and permitting pga to be transcribed more frequently.

In seeking candidates for this repressor, we made the assumption that the enzymatic activity of PGA is functionally linked to PAA metabolism because PGA production is greatly stimulated by PAA. We therefore focused on the regulatory components of the paa operon responsible for catabolizing PAA for use as a sole carbon source (11). Under normal conditions, PaaX, the repressor of the paa operon encoded by paaX, binds to the divergent Pa and Pz promoters to prevent transcription of the meta-cleavage pathway gene cluster. However, when PAA is present, PaaK, the PAA-CoA ligase encoded by paaK, converts PAA to PAA-CoA and this inhibits PaaX from binding to the promoter and initiates expression from the Pa and Pz promoters (12).

Prompted by these findings, we constructed two deletion mutants of E. coli W, {Delta}paaX and {Delta}paaK, and performed PGA assays with these mutants grown in M9 minimal medium containing glycerol but without PAA. Fig. 1 shows that the {Delta}paaX cells had much higher PGA activity than the wild type cells, and the {Delta}paaK cells had lower activity. As a control we showed that the {Delta}paaX cells transformed with pUC-paaX had reduced basal PGA activity (Fig. 1, panel A), and RT-PCR analysis confirmed that pga mRNA was elevated in the {Delta}paaX cells (Fig. 1, panel B). These findings strongly suggested that PaaX was the repressor of pga expression. In these experiments we used endogenous {beta}-galactosidase as an internal control, and we measured the constitutive levels of both its activity and its mRNA (data not shown).

In order to locate precisely the promoter region to which PaaX binds, we generated various promoter deletions by PCR and subcloned them into pUCK. Wild type cells transformed with these plasmids were grown in M9 minimal medium containing glycerol, and their PGA activities were measured (Fig. 2). The results with the first group of plasmids (pUCK-C3, -C4, and -C7) show that the region from -200 to -1 (pUCK-C3) is sufficient for PaaX-binding, i.e. it represents the pga operator, but neither the region from -100 to -1 (pUCK-C4) nor the region from -200 to -101 (pUCK-C7) is sufficient. The results with the second group of plasmids (pUCK-C10, -C11, -C14, and -C15) establish that the operator extends from -120 to -81. The third group of plasmids (pUCK-C16 to -C19) indicates that two regions, Fig. 2, boxed regions labeled A and B, are necessary for PaaX-binding. We cannot, however, rule out the possibility that the sequence or length of the intervening region may also important for binding.



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  		FIG. 2.
In vivo PaaX titration assays with plasmids harboring different upstream regions of the pga promoter. The horizontal lines indicate the promoter regions present in each plasmid. The relative PGA activity (%) of the transformants is given on the right, with activity exceeding the endogenous PGA in boldface. The boxed regions labeled A and B cover from -120 to -111 and from -90 to -81, respectively. All values are averages of three independent experiments, and standard deviations did not exceed 15%. RA, relative activity.

 
To establish directly the identity of the binding sites of PaaX in the pUCK-C1 plasmid, we carried out a DNase I footprinting experiment with purified His-PaaX proteins (Fig. 3). His-PaaX indeed protected only one segment of the pga upstream region, of about 50 bases in length, from -126 to -77, a very unusual upstream binding site for a bacterial repressor. The pga operator defined by this footprinting experiment is virtually identical to the region revealed by the titration assays in Fig. 2. Despite the fact that increasing the concentration of His-PaaX resulted paradoxically in weaker footprint bands, it was clear that there is a hypersensitive site in the center of the protected region. This suggests that PaaX binds to DNA in pairwise fashion, in the form of dimers or tetramers, etc., leaving an unprotected central region exposed to attack by DNase I.

Identification of a New PaaX-binding Consensus Sequence—To determine the precise sequence to which PaaX binds we constructed six plasmids each with a single base substitution in the presumed operator region. In PaaX titration assays using these plasmids, pUCK-CP-84 and -CP-119, with mutations located in the regions shown in Fig. 2, boxed regions A and B, respectively, failed to titrate PaaX (supplemental data 1, Table A). Because a single base substitution in Fig. 2, boxed regions A and B, is enough to abolish operator activity, it seems reasonable to suppose that PaaX binds in a pairwise manner, with one molecule recognizing region A and the other region B. This would also be consistent with the result of the DNase I footprinting experiments.

In order to examine Fig. 2, boxed regions A and B, more closely, we prepared two additional groups of plasmids harboring pga operators with single base substitutions in every other base in those regions. The analysis showed that pUCK-CP-116 and -CP-119 in region A and pUCK-CP-83, -CP-84, and -CP-87 in region B were almost totally ineffective in titrating PaaX (supplemental data 1, Table B). Examination of these essential bases revealed a unique inverted repeat in the pga operator. We subsequently compared the sequences of other PaaX-binding promoters, Pa and Pz in the paa operon, with those of the pga operator, and we found that those promoters also contained this repeat sequence. These findings therefore permit us to identify a new consensus sequence for PaaX (Fig. 4). This consists of a palindromic 6-base sequence separated by 27 intervening bases, TGATTC(N27)GAATCA. One or two bases in each 6-base element of the related promoters deviates from this consensus, as does the length of the intervening sequence. Further analysis of the paaXY promoter of E. coli W and the pga promoter of Kluyvera cryocrescens (46) has shown that this new PaaX-binding consensus sequence also applies to these promoters (Fig. 4).



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  		FIG. 4.
Proposed PaaX-binding consensus sequence. Sequences of the PaaX-binding regions of Pa, Pz, and Ppga promoters. The consensus sequence is made up of a perfect 6-base inverted repeat separated by 27 intervening bases. The double underlined text in the Ppga line depicts the PaaX-binding region of Fig. 2, and asterisks mark the residues implicated by PaaX titration assays in supplemental data 1. The arrows in the line labeled consensus indicate the inverted repeat. Pa is the promoter of the paaABCDEFGHIJK operon, Pz is that of paaZ operon, and Px is that of paaXY in the paa-operon; Ppga(Kluyvera) is that of the pga gene of K. cryocrescens.

 
PAA Relieves PaaX Repression of pga Expression—As stated, PAA-CoA, generated from PAA by PaaK, inhibits PaaX from binding to the Pa and Pz promoters of the paa operon (12). We have confirmed by EMSA that it also inhibits His-PaaX from binding to the pga promoter (supplemental data 2). We therefore hypothesized that the increase in pga expression that occurs in response to exogenous PAA was due to PAA-CoA-mediated relief of pga from repression by PaaX. To prove this hypothesis, we measured PGA activity of wild type E. coli W and its {Delta}paaX and {Delta}paaK deletion derivatives throughout growth in M9 minimal medium containing glycerol with and without PAA.

In wild type cells, pga was constitutively expressed at a basal level in the absence of PAA; in its presence, expression increased 30-fold in early log phase and declined slowly thereafter, with a small peak of activity in late log phase (Fig. 5, panel A). RT-PCR confirmed the correlation between pga transcription and PGA activity; the pga transcript was abundant in early log phase when PAA was present and almost absent in stationary phase, whereas there was scarcely any pga mRNA in cells grown without PAA (Fig. 5, panel D). As in the previous experiments, measurements of endogenous {beta}-galactosidase activity and mRNA, used as an internal control, yielded a uniform pattern of expression (data not shown).

The {Delta}paaX mutant, as expected, had greatly increased PGA activity when grown without PAA, and the pattern of its activity was similar to that of wild type cells grown with PAA (Fig. 5, panel B). Because the major difference between the {Delta}paaX mutant and the wild type is the presence of free PaaX in the latter that can bind to the pga promoter and be released from it by PAA-CoA, we infer that relief from PaaX repression is necessary for efficient expression of pga. This inference is further substantiated by the fact that the {Delta}paaK mutant, in which derepression of pga cannot occur, produces only basal levels of PGA even with PAA present (Fig. 5, panel C). All results including the Supplemental Material suggest that PAA relieves PaaX-dependent repression of pga via PAA-CoA.

The pga Promoter Is a Class III CRP-dependent Promoter—Besides PAA induction, another important feature of pga expression is catabolite repression by metabolic carbohydrates and polyalcohols (10, 22). When we carried out the previous PGA assays in M9 minimal medium containing glucose, the basal level of PGA activity was significantly reduced (Table I). In order to examine whether CRP is involved in this glucose repression, we constructed {Delta}crp and {Delta}cya mutants (where cya adenylate cyclase gene), and we measured their PGA activities during growth in M9 minimal medium containing glycerol. Both of the deletion strains showed almost negligible PGA activities even though they were grown in medium containing glycerol with or without PAA (Table I). This demonstrates that the cAMP-CRP complex is necessary only for activation of pga expression.


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  		TABLE I
PGA assays under conditions of catabolite repression/derepression

 
In order to study in more detail the role of the cAMP-CRP complex in pga expression, we performed EMSAs with the various derivatives of the pga promoter together with purified CRP proteins. When we used the pga promoter of the region from -200 to -1 (probe a), cAMP-CRP formed two binding complexes, demonstrating that both sites of "probe a," site I (from -119 to -110) and site II (from -77 to -57), are actually able to bind CRP in vitro (Fig. 6, panel A). When we used the promoters with the 2-base substitutions in the CRP-binding consensus ("probe b" and "probe d"), mutations in site II completely prevented binding, whereas mutations in site I ("probe c") did not (Fig. 6, panel A). From theses results, we inferred that site II has the greater affinity for cAMP-CRP and that its prior binding facilitates subsequent binding of cAMP-CRP to site I, generating the slowly migrating complex. To prove this fact, we carried out EMSAs with promoters harboring only one of the two CRP-binding sites. As expected, site I (probe e) had much lower affinity than site II (probe f) (Fig. 6, panel B).

We also investigated the function of the two CRP-binding sites in vivo by assaying constructs with {beta}-galactosidase fused to the pga promoters used in the EMSAs. We constructed a {Delta}lacZ mutant of E. coli W, transformed it with each of the fusion constructs, and measured {beta}-galactosidase activity during growth in M9 minimal medium containing glycerol (Table II). The construct with a wild type pga promoter had full activity and acted like the endogenous pga promoter because {beta}-galactosidase activity was elevated about 3-fold when PAA was present. On the other hand, all the fusion constructs with mutant pga promoters had less than half the basal activity of the wild type promoter, showing that CRP bindings to both sites are required for full pga expression.


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  		TABLE II
{beta}-Galactosidase assays with lacZ constructs fused with pga promoters with various mutations

 
From the results of the in vivo and in vitro experiments, we can conclude that the pga promoter is a class III CRP-dependent promoter (47), in which site II has much higher affinity for cAMP-CRP, and that the subsequent binding of a complex to site I is necessary for efficient transcription. This interpretation accounts well for the fact that the pga promoter is barely active when only one site is occupied by cAMP-CRP (pMAK705-site I in Table II). In addition, in view of the fact that the PaaX operator (from -126 to -77) completely overlaps the weaker CRP-binding site (site I from -119 to -100), we would anticipate that competition between PaaX and cAMP-CRP for binding to the pga upstream region would influence pga expression.

Regulation of pga Expression by PaaX and the cAMP-CRP Complex—To demonstrate antagonistic regulation of pga expression by PaaX as repressor, and CRP as activator, we constructed 3'-cMyc-tagged derivatives of recombinant paaX and crp and performed ChIP assays. Fig. 7, panel A, shows that PaaX binds very tightly to the pga promoter in the absence of PAA but fails to bind in its presence and that, conversely, the cAMP-CRP complex binds strongly to the pga promoter in the presence of PAA but hardly at all in its absence. These findings clearly support the idea that these factors compete for binding to the pga promoter and that the outcome depends on whether PAA is present or not.



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  		FIG. 7.
Mutually exclusive binding of PaaX and cAMP-CRP to the pga promoter. {Delta}paaX and {Delta}crp cells were transformed with pREP42-paaX and pREP42-crp, respectively. Chromosomal DNA was prepared from the transformants at A600 {approx} 0.5, and co-immunoprecipitation (IP) and PCR were carried out as described under "Experimental Procedures." The {Delta}lacZ cells carrying the pMAK705 derivatives used in Table II were transformed with pREP42-crp, and the procedures were carried out as previously. Two independent assays yielded similar results, and a typical one is shown here. Panel A, competitive binding of PaaX and CRP to the pga promoter according to the presence or absence of PAA. Panel B, binding of CRP complex to the pga promoter containing the mutated CRP-binding sites. WT, wild type.

 
In view of the fact that the operator for PaaX covers only the CRP-binding site I, cAMP-CRP could conceivably bind to site II when PAA is absent. This would account for the observation that cAMP-CRP bound to the pga promoter harboring mutations in site I (Fig. 7, panel B). Moreover, these assays confirm that site II has a higher affinity than site I in vivo, as no complex formed using promoters with base substitutions in site II, whereas some complex formed with promoters with mutations in site I. This is consistent with the results of the EMSAs (Fig. 6, panel A).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have established some features of the regulation of pga expression. First, we have demonstrated that PaaX represses pga transcription, and this accounts both for negative regulation and PAA induction of pga expression. In connection with the negative regulation of pga, it was proposed previously that PacR was the repressor and, moreover, the component responsible for PAA induction (15, 48). However, we believe this to be unlikely for several reasons (see also Ref. 10). Thus, although the pacR gene is located inside the pga structural gene (49), translational fusions (Ppga::lacZ and Ppga::phoA) that lack the pga structural gene continue to be inducible by PAA (20, 50). Moreover, when we measured PGA activity in E. coli W cells transformed with plasmids expressing the pacR gene, we found normal levels of PGA activity rather than reduced levels as expected if PacR is a repressor (data not shown). Thus, since we have shown here that PAA induction consists primarily of relief of pga from PaaX repression by PAA, we suggest that the increase in expression of pga in response to PAA be renamed "PAA derepression." Our findings therefore provide answers to some of the questions posed by Diaz et al. (10) concerning the regulatory proteins involved in PAA induction; PaaX is responsible for repression of pga expression and PAA for derepression.

We believe that we have also identified the consensus sequence recognized by PaaX. This consensus is totally different from the earlier proposal of a 15-base imperfect palindromic motif restricted to the left half of the operators of the Pa and Pz promoters (12). We believe this previous suggestion to be incorrect for the following reasons. (i) The {beta}-galactosidase assays and EMSAs with point mutant promoters carried out by Ferrandez et al. (12) were restricted to the left half of the PaaX-binding region. (ii) The sequence analysis concentrated on the "TGTGA" motif and did not consider other potential sequences. (iii) The hypersensitive sites detected by DNase I footprinting were located centrally, indicating that the right half ought to play some role in PaaX binding. (iv) In the present work we did not observe the 15-base PaaX-binding motif of Ferrandez et al. (12) in either the left or right half of the pga operator. Reevaluation of the previous results obtained with the Pa and Pz promoters actually supports the consensus sequence that we propose. First, Ferrandez et al. (12) observed that mutations within our 6-base element reduced PaaX binding and resulted in less repression in the {beta}-galactosidase assay, and the effect of 2-base substitutions within the 6-base element was greater than that of 1-base substitutions (pAFPA2-M1, -M5, -M9, and -M16 in Fig. 5 and Table III of Ref. 12). Second, single base deletions in what we now consider the intervening sequence had a clear-cut effect on binding and repression (pAFPA2-M18 and -M22 in Fig. 5 and Table III of Ref. 12). Third, the lower PaaX-binding affinity observed for Pz can be accounted for by the presence of an additional base within the intervening sequence (Fig. 1 of Ref. 12). Finally, the finding by Ferrandez et al. (12) of central hypersensitive bases in DNase I footprinting assays (Fig. 3 of Ref. 12) strongly supports our proposal of pairwise binding of PaaX to the 6-base element in each half of the protected region. These findings indicate that not only the two 6-base elements but also the distance between them are important, and that base changes, insertions, and deletions can cause PaaX to bind more loosely and consequently to repress its target genes less efficiently.

PaaX binds to an unusual, upstream, region in the pga promoter, and it does not seem to have an effect on RNA polymerase but instead on the cAMP-CRP activator. However, it binds to the Pa promoter in the region immediately after the -10 box and to Pz promoter in the region overlapping the -10 box, and therefore would interrupt the binding or procession of RNA polymerase in those instances (12). This diversity in the mechanism of transcriptional repression by PaaX is noteworthy because it acts as an upstream repressor and functions by displacing the activator, cAMP-CRP, in the pga promoter, unlike most bacterial repressors that inhibit either RNA polymerase, open-complex formation, or transcriptional activation by binding close to the transcription initiation site. In addition, although pga expression must be controlled coordinately with the paa operon, by repression by PaaX and derepression by PAA-CoA, the fine regulation of pga transcription involving the cAMP-CRP complex is quite different from that of the paa operon.2

The earlier suggestion that CRP-binding site I is the major binding site (21) was based on measurements of Ppga::lacZ fusion constructs containing either point mutations in site I or 5-base deletions that placed site I on the opposite side of the DNA helix, as well as on structural modeling of mutant pga promoters. However, those studies did not include any investigation of site II, and therefore the relative importance of the two CRP-binding sites could not be properly evaluated. Since the results of Stojcevic et al. (21) are well accounted for by our model and, moreover, we provide evidence that the cAMP-CRP complex binds directly to the pga promoter, it seems clear that it is the CRP-binding site II that has the higher affinity for cAMP-CRP.

Recently, the acsP2 promoter was reported to be the first example of a native class III CRP promoter (51). In the acsP2 promoter, the upstream CRP site II centered at position -122.5 has low affinity and CRP that is bound there acts as a coactivator for optimal activation. This is partially consistent with our results; the upstream pga CRP site I centered at -109.5 has the lower affinity. It was also reported that the nucleoproteins, FIS and IHF, bound directly to multiple regions of the acsP2 promoter, that their binding reduced CRP-dependent acs transcription, and that FIS competed with CRP to occupy the acsP2 promoter. This regulation mechanism is very similar to that of pga in view of competitive binding (52). However, there are some differences between the two situations. Thus (i) base substitutions at either site I or II of the pga promoter permit similarly negligible levels of pga expression (Table II), whereas substitutions at upstream site II of the acs promoter had moderate decrease of its expression. (ii) Although base substitutions at the downstream site I of the acs promoter prevented CRP binding and CRP-dependent activation, those at the upstream site I of the pga promoter permitted CRP binding but rendered CRP unable to activate transcription (Fig. 6 and Table II). (iii) Most important, the lower affinity site I of the pga promoter is overlapped with binding site of the PaaX repressor; hence it acts not only as a co-activating site but also as a major regulatory site.

From the results obtained in this work, we can now describe the regulation of pga expression as represented schematically in Fig. 8. During normal growth of E. coli W, PaaX is expressed and binds to its cognate region on the pga operator. In the absence of PAA, pga is poorly expressed because the cAMP-CRP complex cannot bind to the CRP-binding site I as the latter is occupied by PaaX. When PAA is supplied it enters the nucleoplasm and is converted to PAA-CoA by PaaK, and this then inhibits PaaX from binding to the pga operator by blocking the physical interaction between PaaX monomers.2 The cAMP-CRP complex is thus now able to bind to the unoccupied CRP-binding site I after binding to the higher affinity CRP-binding site II, and then fully activates pga transcription in the manner typical of class III CRP-dependent promoter (Fig. 8).



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  		FIG. 8.
Model of the overall regulation of pga expression. In this model the transcriptional repressor, PaaX, and the transcriptional activator, the cAMP-CRP complex, compete for binding to the upstream region of the pga promoter that overlaps with the CRP-binding site I. Details are given in the text. Panel A, schematic representation of the pga promoter with the PaaX-binding region, the pga operator, indicated by a dotted oval. Panel B, PaaX repression of pga during normal growth. Panel C, derepression of pga in the presence of PAA. The pair of PaaX molecules in panels B and C is not necessarily meant to imply that they dimerize but only that they interact.

 

   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains Data 1 and 2 and Tables A and B. Back

{ddagger} Present address: CJ Corp., R & D Center of Bioproducts, San 522-1, Dokpyong-Ri, Majang-Myon, Ichon-Si, Kyonggi-Do, 467-812, Korea. Back

§ Supported by BK21 Research Fellowship from the Ministry of Education and Human Resources Development. Back

To whom correspondence should be addressed. Tel.: 82-2-880-6701; Fax: 82-2-876-4440; E-mail: khslab{at}snu.ac.kr.

1 The abbreviations used are: PGA, penicillin G acylase; 6-APA, 6-aminopenicillanic acid; PAA, phenylacetic acid; PAA-CoA, phenylacetic acid-coenzyme A; CRP, cAMP-receptor protein; IHF, integration host factor; RT, reverse transcriptase; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation. Back

2 H. S. Kim, T. S. Kang, and H. S. Kang, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Deog Su Hwang and Julian Gross for helpful discussions and critical advice on the manuscript.



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