Regulation of Penicillin G Acylase Gene Expression in Escherichia coli by Repressor PaaX and the cAMP-cAMP Receptor Protein Complex
2004; Elsevier BV; Volume: 279; Issue: 32 Linguagem: Inglês
10.1074/jbc.m404348200
ISSN1083-351X
AutoresHyoung Seok Kim, Tae Sun Kang, Joon Sik Hyun, Hyen Sam Kang,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe 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. 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. Penicillin G acylase (PGA, 1The 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. EC 3.5.1.11) is a type II penicillin acylase that hydrolyzes penicillin G to 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA) (1Virden R. Biotechnol. & Genet. Eng. Rev. 1990; 8: 189-218Crossref Scopus (23) Google Scholar). It is one of the most important industrial enzymes for the production of semi-synthetic penicillins (2Valle F. Balbas P. Merino E. Bollvar F. Trends Biochem. Sci. 1991; 16: 36-40Abstract Full Text PDF PubMed Scopus (107) Google Scholar). PGAs have been found in numerous bacteria and fungi (3Martin L. Prieto M.A. Cortes E. Garcia J.L. FEMS Microbiol. Lett. 1995; 125: 287-292Crossref PubMed Scopus (42) Google Scholar, 4Barbero J.L. Buesa J.M. Buitrago G.G. Mendez E. Aranda A.P. Garcia J.L. Gene (Amst.). 1986; 49: 69-80Crossref PubMed Scopus (75) Google Scholar, 5Verhaert R.M.D. Riemens A.M. Laan A.M. Duin J. Quax W.J. Appl. Environ. Microbiol. 1997; 63: 3412-3418Crossref PubMed Google Scholar, 6Ohashi H. Katsuta Y. Hashizume T. Abe S.N. Kajiura H. Hattori H. Kamei T. Yano M. Appl. Environ. Microbiol. 1988; 54: 2603-2607Crossref PubMed Google Scholar, 7Mahmood Z.A. Shaikh D. Zoha S.M. Folia Microbiol. 1991; 36: 444-446Crossref PubMed Scopus (1) Google Scholar, 8Ljubijankic G. Konstantinovic M. Gilson V. DNA Seq. 1992; 3: 195-200Crossref PubMed Scopus (24) Google Scholar), and the PGA of Escherichia coli W ATCC11105 is the best characterized (2Valle F. Balbas P. Merino E. Bollvar F. Trends Biochem. Sci. 1991; 16: 36-40Abstract Full Text PDF PubMed Scopus (107) Google Scholar, 9Sudhakaran V.K. Deshpande B.S. Ambedkar S.S. Shewale J.G. Process Biochem. 1992; 27: 131-143Crossref Scopus (45) Google Scholar). 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 (2Valle F. Balbas P. Merino E. Bollvar F. Trends Biochem. Sci. 1991; 16: 36-40Abstract Full Text PDF PubMed Scopus (107) Google Scholar, 10Diaz E. Ferrandez A. Prieto M.A. Garcia J.L. Microbiol. Mol. Biol. Rev. 2001; 65: 523-569Crossref PubMed Scopus (289) Google Scholar). 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 (11Ferrandez A. Minambres B. Garcia B. Olivera E.R. Luengo J.M. Garcia J.L. Diaz E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), which in turn inhibits PaaX, the repressor of the paa operon, from binding to DNA (12Ferrandez A. Garcia J.L. Diaz E. J. Biol. Chem. 2000; 275: 12214-12222Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). As a consequence the paa-operon is expressed, and the meta-cleavage pathway subsequently degrades PAA-CoA before it enters the tricarboxylic acid cycle (13Luengo J.M. Garcia J.L. Olivera E.R. Mol. Microbiol. 2001; 39: 1434-1442Crossref PubMed Scopus (127) Google Scholar). The expression of E. coli pga is regulated by many factors such as temperature (14Oh S.J. Kim Y.C. Park Y.W. Min S.Y. Kim I.S. Kang H.S. Gene (Amst.). 1987; 56: 87-97Crossref PubMed Scopus (57) Google Scholar, 15Dai M. Zhu Y. Yang Y. Wang E. Xie Y. Zhao G. Jiang W. Eur. J. Biochem. 2001; 268: 1298-1303Crossref PubMed Scopus (15) Google Scholar), oxygen (16Vojtisek V. Slezak J. Folia Microbiol. 1975; 20: 298-306Crossref PubMed Scopus (17) Google Scholar, 17Gang D.M. Shaikh K. Biochim. Biophys. Acta. 1976; 425: 110-114Crossref PubMed Scopus (38) Google Scholar, 18Yang Y.G. Xu J.N. Hu T.S. Qian Y.C. Yang S.L. Gong Y. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2000; 32: 163-168PubMed Google Scholar), carbon source (19Merino E. Balbas P. Recillas F. Becerril B. Valle F. Bolivar F. Mol. Microbiol. 1992; 6: 2175-2182Crossref PubMed Scopus (60) Google Scholar, 20Roa A. Garcia J.L. FEMS Microbiol. Lett. 1999; 177: 7-14Crossref PubMed Google Scholar, 21Stojcevic N. Moric I. Begovic J. Radoja S. Konstantinovic M. Biomol. Eng. 2001; 17: 113-117Crossref PubMed Scopus (5) Google Scholar), and PAA (22Szentimai A. Appl. Microbiol. 1964; 12: 185-187Crossref PubMed Google Scholar). It is also controlled at the level of translocation (18Yang Y.G. Xu J.N. Hu T.S. Qian Y.C. Yang S.L. Gong Y. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2000; 32: 163-168PubMed Google Scholar, 23Scherrer S. Robas N. Zouheiry H. Branlant G. Branlant C. Appl. Microbiol. Biotechnol. 1994; 42: 85-91Crossref PubMed Scopus (28) Google Scholar, 24Ignatova Z. Hornle C. Nurk A. Kasche V. Biochem. Biophys. Res. Commun. 2002; 291: 146-149Crossref PubMed Scopus (69) Google Scholar), auto-processing (25Choi K.S. Kim J.A. Kang H.S. J. Bacteriol. 1992; 174: 6270-6276Crossref PubMed Google Scholar, 26Duggleby H.J. Tolley S.P. Hill C.P. Dodson E.J. Dodson G. Moody P.C.E. Nature. 1995; 373: 264-268Crossref PubMed Scopus (424) Google Scholar, 27Kasche V. Lummer K. Nurk A. Piotraschke E. Rieks A. Stoeva S. Voelter W. Biochim. Biophys. Acta. 1999; 1433: 76-86Crossref PubMed Scopus (68) Google Scholar), and stabilization by chaperons (28Chou C.P. Tseng J.H. Kuo B.Y. Lai K.M. Lin M.I. Lin H.K. Biotechnol. Prog. 1999; 15: 439-445Crossref PubMed Scopus (20) Google Scholar, 29Lin W.J. Huang S.W. Chou C.P. Biotechnol. Bioeng. 2001; 73: 484-492Crossref PubMed Scopus (24) Google Scholar, 30Pan K.L. Hsiao H.C. Weng C.L. Wu M.S. Chou C.P. J. Bacteriol. 2003; 185: 3020-3030Crossref PubMed Scopus (43) Google Scholar). E. coli PGA is the first example of auto-proteolytic processing of an inactive precursor to a functional protein in bacteria (31Meyer T.L. Bock A. Hennecke H. FEBS Lett. 1992; 307: 62-65Crossref PubMed Scopus (17) Google Scholar), and this occurs in a pH- and temperature-dependent manner (15Dai M. Zhu Y. Yang Y. Wang E. Xie Y. Zhao G. Jiang W. Eur. J. Biochem. 2001; 268: 1298-1303Crossref PubMed Scopus (15) Google Scholar, 32Lee H.S. Park O.K. Kang H.S. Biochem. Biophys. Res. Commun. 2000; 272: 199-204Crossref PubMed Scopus (11) Google Scholar). According to the x-ray crystallographic analysis (26Duggleby H.J. Tolley S.P. Hill C.P. Dodson E.J. Dodson G. Moody P.C.E. Nature. 1995; 373: 264-268Crossref PubMed Scopus (424) Google Scholar), PGA belongs to the family of N-terminal nucleophile hydrolases (33Oinonen C. Rouvinen J. Protein Sci. 2000; 9: 2329-2337Crossref PubMed Scopus (209) Google Scholar). Previous studies of the E. coli pga promoter identified two putative CRP-binding sites (14Oh S.J. Kim Y.C. Park Y.W. Min S.Y. Kim I.S. Kang H.S. Gene (Amst.). 1987; 56: 87-97Crossref PubMed Scopus (57) Google Scholar, 34Valle F. Gosset G. Tenorio B. Oliver G. Bolivar F. Gene (Amst.). 1986; 50: 119-122Crossref PubMed Scopus (31) Google Scholar) and two possible IHF-binding sites (21Stojcevic N. Moric I. Begovic J. Radoja S. Konstantinovic M. Biomol. Eng. 2001; 17: 113-117Crossref PubMed Scopus (5) Google Scholar); however, there is no direct evidence that CRP or IHF proteins bind to the promoter (35Werner M.H. Burley S.K. Cell. 1997; 88: 733-736Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). It has been inferred that the pga promoter is a class III CRP-dependent promoter (21Stojcevic N. Moric I. Begovic J. Radoja S. Konstantinovic M. Biomol. Eng. 2001; 17: 113-117Crossref PubMed Scopus (5) Google Scholar). 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. 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 β-lactamase gene of pUC18 (36, Kim, H. S. (1995) Analysis of regulatory sequences and factor involved in the expression of penicillin G acylase gene in Escherichia coli. M.Sc. thesis, Seoul National UniversityGoogle Scholar). 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 (37Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar) 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 (38Sambrook J. Russel D. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar) by using E. coli DH5α cells grown at 37 °C with shaking at 200 rpm in LB medium containing 0.5 mm isopropyl-1-thio-β-d-galactopyranoside and 40 μg/ml 5-bromo-4-chloro-3-indolyl-β-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 (39Simons R.W. Houman F. Kleckner N. Gene (Amst.). 1987; 53: 85-96Crossref PubMed Scopus (1301) Google Scholar) 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 (40Hamilton C.M. Aldea M. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar) 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 λ RED system (41Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar). E. coli W ATCC11105 cells were transformed with pIJ790 (42Gust B. Challis G.L. Fowler K. Kieser T. Chater K.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1541-1546Crossref PubMed Scopus (1222) Google Scholar), and electrocompetent cells were prepared as described (41Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar) with some modifications. (i) The cells were cultured in LB medium. (ii) l-Arabinose was used to induce the λ 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 (32Lee H.S. Park O.K. Kang H.S. Biochem. Biophys. Res. Commun. 2000; 272: 199-204Crossref PubMed Scopus (11) Google Scholar, 43Bomstein J. Evans W.G. Anal. Chem. 1965; 37: 576-578Crossref PubMed Scopus (58) Google Scholar). β-Galactosidase Assays—β-Galactosidase activities of E. coli W, grown at 30 °C on a shaking incubator, were measured as described (37Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar). 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.Fig. 1Evidence that PaaX is the transcriptional repressor of pga expression.Panel A, PGA activity of wild type (WT), ΔpaaX, ΔpaaK, and ΔpaaX 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, ΔpaaX, and ΔpaaK cells at A600 ≈ 0.2. Three independent experiments gave similar results, and a typical one is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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-β-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 [α-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 0.5× 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. 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. Chromatin Immunoprecipitation (ChIP) Assays—The chromatin immunoprecipitation assays for detecting in vivo protein-DNA interactions were done essentially as described (44Hecht A. Grunstein M. Methods Enzymol. 1999; 304: 399-414Crossref PubMed Scopus (151) Google Scholar) with minor modifications. pREP42-paaX and pREP42-crp were transformed with the ΔpaaX and Δcrp, 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 2× 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. PaaX Is the Repressor of pga Expression—Although E. coli pga has been known for a long time to be negatively regulated (45Mayer H. Collins J. Wagner F. Timmis K.N. Puhler A. Plasmids of Medical, Environmental, and Commercial Importance. Elsevier Science Publishers B.V., Amsterdam1979: 459-470Google Scholar), 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, Kim, H. S. (1995) Analysis of regulatory sequences and factor involved in the expression of penicillin G acylase gene in Escherichia coli. M.Sc. thesis, Seoul National UniversityGoogle Scholar). 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 (11Ferrandez A. Minambres B. Garcia B. Olivera E.R. Luengo J.M. Garcia J.L. Diaz E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). 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 (12Ferrandez A. Garcia J.L. Diaz E. J. Biol. Chem. 2000; 275: 12214-12222Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Prompted by these findings, we constructed two deletion mutants of E. coli W, ΔpaaX and ΔpaaK, and performed PGA assays with these mutants grown in M9 minimal medium containing glycerol but without PAA. Fig. 1 shows that the ΔpaaX cells had much higher PGA activity than the wild type cells, and the ΔpaaK cells had lower activity. As a control we showed that the ΔpaaX 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 ΔpaaX cells (Fig. 1, panel B). These findings strongly suggested that PaaX was the repressor of pga expression. In these experiments we used endogenous β-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 gene
Referência(s)