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• W2035940253 abstract "In Escherichia coli there are two pathways for conversion of adenine into guanine nucleotides, both involving the intermediary formation of IMP. The major pathway involves conversion of adenine into hypoxanthine in three steps via adenosine and inosine, with subsequent phosphoribosylation of hypoxanthine to IMP. The minor pathway involves formation of ATP, which is converted via the histidine pathway to the purine intermediate 5-amino-4-imidazolecarboxamide ribonucleotide and, subsequently, to IMP. Here we describe E. coli mutants, in which a third pathway for conversion of adenine to IMP has been activated. This pathway was shown to involve direct deamination of adenine to hypoxanthine by a manganese-dependent adenine deaminase encoded by a cryptic gene, yicP, which we propose be renamed ade. Insertion elements, located from −145 to +13 bp relative to the transcription start site, activated theade gene as did unlinked mutations in the hnsgene, encoding the histone-like protein H-NS. Gene fusion analysis indicated that ade transcription is repressed more than 10-fold by H-NS and that a region of 231 bp including theade promoter is sufficient for this regulation. The activating insertion elements essentially eliminated the H-NS-mediated silencing, and stimulated ade gene expression 2–3-fold independently of the H-NS protein. In Escherichia coli there are two pathways for conversion of adenine into guanine nucleotides, both involving the intermediary formation of IMP. The major pathway involves conversion of adenine into hypoxanthine in three steps via adenosine and inosine, with subsequent phosphoribosylation of hypoxanthine to IMP. The minor pathway involves formation of ATP, which is converted via the histidine pathway to the purine intermediate 5-amino-4-imidazolecarboxamide ribonucleotide and, subsequently, to IMP. Here we describe E. coli mutants, in which a third pathway for conversion of adenine to IMP has been activated. This pathway was shown to involve direct deamination of adenine to hypoxanthine by a manganese-dependent adenine deaminase encoded by a cryptic gene, yicP, which we propose be renamed ade. Insertion elements, located from −145 to +13 bp relative to the transcription start site, activated theade gene as did unlinked mutations in the hnsgene, encoding the histone-like protein H-NS. Gene fusion analysis indicated that ade transcription is repressed more than 10-fold by H-NS and that a region of 231 bp including theade promoter is sufficient for this regulation. The activating insertion elements essentially eliminated the H-NS-mediated silencing, and stimulated ade gene expression 2–3-fold independently of the H-NS protein. insertion elements cAMP receptor protein Escherichia coli auxotrophic mutants can utilize adenine as the sole source of purines. Conversion of adenine into guanine nucleotides occurs by two different pathways that converge on IMP and utilize the subsequent reactions of the de novosynthesis pathway to guanine nucleotides (1Zalkin H. Nygaard P. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology 1. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 561-579Google Scholar). The major pathway involves conversion of adenine to hypoxanthine in three steps involving the intermediate formation of adenosine and inosine (Fig.1). The first and third reactions in this sequence are catalyzed by the deoD gene product, purine nucleoside phosphorylase, whereas the second step is catalyzed by adenosine deaminase encoded by the add gene. Hypoxanthine in turn is converted to IMP by hypoxanthine or guanine phosphoribosyltransferases encoded by the hpt and gpt genes, respectively. The minor pathway involves formation of ATP, which is converted via the histidine pathway to the purine intermediate 5-amino-4-imidazolecarboxamide ribonucleotide and subsequently to IMP (Fig. 1). The flux through this pathway is limited because the first enzyme of the histidine pathway, HisG, is subject to strong feedback inhibition by the end product histidine (2Winkler M.E. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology 1. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 485-505Google Scholar). Thus, purine-requiring mutants in which the major pathway is blocked by mutation of thedeoD gene only grow very slowly with adenine as the sole source of purines, and this residual growth can be eliminated by the addition of histidine to the growth medium (3Neuhard J. Nygaard P. Ingraham J.L. Low K.B. Magasanik B. Neidhardt F.C. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1987: 445-473Google Scholar). These findings indicate that there are no other pathways for converting adenine into guanine nucleotides. Specifically, studies of enzymatic activities in crude extracts indicated that E. coli contains no adenine deaminase activity, which might convert adenine directly to hypoxanthine by deamination (4Leung H.B. Schramm V.L. J. Biol. Chem. 1980; 255: 10867-10874Abstract Full Text PDF PubMed Google Scholar). Nevertheless, Kocharyan et al. (5Kocharyan S.M. Kocharyan A.M. Meliksetyan G.O. Akopyan Z.I. Genetika. 1982; 18: 906-914PubMed Google Scholar) reported the isolation of E. coli mutants, in which an apparently cryptic adenine deaminase gene had been activated. The genetic locus affected in these mutants, however, was not identified nor mapped. The paradigm example of cryptic genes in E. coli is thebgl operon involved in metabolism of aromatic β-glucosides. A key element in the silencing of the bgloperon is the small abundant nucleoid-associated protein, H-NS, which probably forms a repressing nucleoprotein complex upon binding to silencer DNA regions flanking the bgl promoter (6Reynolds A.E. Mahadevan S., Le Grice S.F. Wright A. J. Mol. Biol. 1986; 191: 85-95Crossref PubMed Scopus (83) Google Scholar, 7Higgins C.F. Dorman C.J. Stirling D.A. Waddell L. Booth I.R. May G. Bremer E. Cell. 1988; 52: 569-584Abstract Full Text PDF PubMed Scopus (513) Google Scholar, 8Schnetz K. EMBO J. 1995; 14: 2545-2550Crossref PubMed Scopus (68) Google Scholar, 9Mukerji M. Mahadevan S. Mol. Microbiol. 1997; 24: 617-627Crossref PubMed Scopus (60) Google Scholar, 10Caramel A. Schnetz K. J. Mol. Biol. 1998; 284: 875-883Crossref PubMed Scopus (57) Google Scholar). In addition to the bgl operon and other cryptic genes the H-NS protein also modulates the expression of a large number of activeE. coli genes, usually by repressing transcription initiation (11Atlung T. Ingmer H. Mol. Microbiol. 1997; 24: 7-17Crossref PubMed Scopus (401) Google Scholar, 12Hommais F. Krin E. Laurent-Winter C. Soutourina O. Malpertuy A., Le Caer J. Danchin A. Bertin P. Mol. Microbiol. 2001; 40: 20-36Crossref PubMed Scopus (342) Google Scholar). H-NS binds to DNA with no obvious sequence specificity, but specific binding sites tend to be AT-rich and intrinsically bend (13Tanaka K. Muramatsu S. Yamada H. Mizuno T. Mol. Gen. Genet. 1991; 226: 367-376Crossref PubMed Scopus (85) Google Scholar, 14Owen-Hughes T.A. Pavitt G.D. Santos D.S. Sidebotham J.M. Hulton C.S. Hinton J.C.D. Higgins C.F. Cell. 1992; 71: 255-265Abstract Full Text PDF PubMed Scopus (245) Google Scholar, 15Lucht J.M. Dersch P. Kempf B. Bremer E. J. Biol. Chem. 1994; 269: 6578-6586Abstract Full Text PDF PubMed Google Scholar, 16Zuber F. Kotlarz D. Rimsky S. Buc H. Mol. Microbiol. 1994; 12: 231-240Crossref PubMed Scopus (72) Google Scholar), as also observed for the upstream silencing region of the bgl operon (6Reynolds A.E. Mahadevan S., Le Grice S.F. Wright A. J. Mol. Biol. 1986; 191: 85-95Crossref PubMed Scopus (83) Google Scholar, 8Schnetz K. EMBO J. 1995; 14: 2545-2550Crossref PubMed Scopus (68) Google Scholar, 17Timchenko T. Bailone A. Devoret R. EMBO J. 1996; 15: 3986-3992Crossref PubMed Scopus (45) Google Scholar). Binding of H-NS generally induces strong condensation of DNA, and thus, the protein has been implicated in the organization and compaction of the bacterial nucleoid (18Spassky A. Rimsky S. Garreau H. Buc H. Nucleic Acids Res. 1984; 12: 5321-5340Crossref PubMed Scopus (177) Google Scholar). H-NS consists of an N-terminal oligomerization domain and a C-terminal DNA binding domain, and the ability of the protein to condense DNA and repress transcription apparently depends on its ability to oligomerize (19Ueguchi C. Suzuki T. Yoshida T. Tanaka K. Mizuno T. J. Mol. Biol. 1996; 263: 149-162Crossref PubMed Scopus (135) Google Scholar, 20Spurio R. Falconi M. Brandi A. Pon C.L. Gualerzi C.O. EMBO J. 1997; 16: 1795-1805Crossref PubMed Scopus (162) Google Scholar, 21Smyth C.P. Lundback T. Renzoni D. Siligardi G. Beavil R. Layton M. Sidebotham J.M. Hinton J.C. Driscoll P.C. Higgins C.F. Ladbury J.E. Mol. Microbiol. 2000; 36: 962-972Crossref PubMed Scopus (89) Google Scholar). In the present work we have isolated and characterized mutants with increased adenine deaminase activity and demonstrate that this activity is due to a manganese-dependent adenine deaminase encoded by a cryptic gene, yicP, which we propose be renamedade. In agreement with these findings, purified YicP protein has recently been shown to posses significant adenine deaminase activity (22Matsui H. Shimaoka M. Kawasaki H. Takenaka Y. Kurahashi O. Biosci. Biotechnol. Biochem. 2001; 65: 1112-1118Crossref PubMed Scopus (24) Google Scholar). To define the elements responsible for the cryptic nature of the ade gene, we have identified a large number of cis- and trans-acting mutations that lead to activation of gene expression. Like the bgl promoter, the adepromoter region was found to be extremely AT-rich and subject to strong repression by the H-NS protein. As also observed for the bgloperon, we found that insertion of a variety of IS elements within an extended region surrounding the ade promoter resulted in relief of the H-NS-mediated silencing. The results suggest that these IS1elements interfered with the formation of an H-NS·DNA complex, which would otherwise sequester the adjacent ade promoter region. The bacterial strains used in this study are all derivatives of E. coliK12 and are listed in Table I. Generalized transductions with lysates of bacteriophage P1vir were performed as described (26Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar). Minimal medium plates contained AB minimal medium (27Clark B. Maaløe O. J. Mol. Biol. 1967; 23: 99-112Crossref Scopus (706) Google Scholar) solidified with 2% of Difco Bacto agar and supplemented with 0.2% glucose or glycerol as the carbon source, 1 μg/ml thiamine, 15 μg/ml nucleobases, 30 μg/ml nucleosides, or 40 μg/ml methionine and histidine when required. The rich medium was Luria broth (LB). Liquid cultures were grown in glucose minimal medium supplemented with 15 μg/ml hypoxanthine and 0.3% casamino acids unless otherwise indicated.Table IBacterial strainsStrain1-aOriginal strain designations are given in parenthesis.Genotype1-b[SØ003]: F−λ−metB rpsL relA spoT supF lamB lonam.Reference/Source/Construction1-cUnselected markers are shown in parenthesis. Resistance to tetracycline, chloramphenicol, or kanamycin was scored on LB medium containing 15, 20, or 25 μg/ml, respectively, of the antibiotics.CN1927 (NK5526)hisG213::Tn10 IN(rrnD-rrnE)1N. Kleckner via CGSC1-dColi Genetic Stock Center, Yale University, New Haven, CT.CN1980[SØ003]purE deoD gsk::kan(23Petersen C. Møller L.B. J. Biol. Chem. 2001; 276: 884-894Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar)CN2349F−araΔ(codB-lac)3 thi recA56(24Petersen C. Møller L.B. Gene (Amst.). 2000; 261: 289-298Crossref PubMed Scopus (90) Google Scholar)CN2388–2[SØ003]purE deoD gsk yicO-2::IS1Growth on adenine as a purine sourceCN2388–6[SØ003]purE deoD gsk yicO-6::IS4Growth on adenine as a purine sourceCN2388–15[SØ003]purE deoD gsk yicO-15::IS1Growth on adenine as a purine sourceCN2388–11[SØ003]purE deoD gsk hns-11::IS1Growth on adenine as a purine sourceCN2388–16[SØ003]purE deoD gsk hns-16::IS1Growth on adenine as a purine sourceCN2451[CN2388–2]lamB+ade::camSee “Experimental Procedures”CN2470 (GM230)hns205::Tn10 and other markers (SupFwt)(7Higgins C.F. Dorman C.J. Stirling D.A. Waddell L. Booth I.R. May G. Bremer E. Cell. 1988; 52: 569-584Abstract Full Text PDF PubMed Scopus (513) Google Scholar)1-ehns205::Tn10 was originally designated osmZ205::Tn10.CN2479[CN1980]hns205::Tn10CN1980 × P1(CN2470), Tetr(SupF)CN2481[CN2388–2]ade::camCN2388–2 × P1(CN2451), CamrCN2498[CN2349]hns205::Tn101-fUsing P1(CN2470), hns205::Tn10 was transduced into CN2349, which was transiently transformed with an unstable plasmid, pMAS53, containing a functional recA gene and a temperature-sensitive replicon (25).CN2588[CN2388–2]hns205::Tn10CN2388–2 × P1(CN2479), TetrCN2589[CN2388–6]hns205::Tn10CN2388–6 × P1(CN2479), TetrCN2590[CN2388–15]hns205::Tn10CN2388–15 × P1(CN2479), Tetr1-a Original strain designations are given in parenthesis.1-b [SØ003]: F−λ−metB rpsL relA spoT supF lamB lonam.1-c Unselected markers are shown in parenthesis. Resistance to tetracycline, chloramphenicol, or kanamycin was scored on LB medium containing 15, 20, or 25 μg/ml, respectively, of the antibiotics.1-d Coli Genetic Stock Center, Yale University, New Haven, CT.1-e hns205::Tn10 was originally designated osmZ205::Tn10.1-f Using P1(CN2470), hns205::Tn10 was transduced into CN2349, which was transiently transformed with an unstable plasmid, pMAS53, containing a functional recA gene and a temperature-sensitive replicon (25Krüger M.K. Pedersen S. Hagervall T.G. Sørensen M.A. J. Mol. Biol. 1998; 284: 621-631Crossref PubMed Scopus (105) Google Scholar). Open table in a new tab An auxotrophic purE deoD strain, CN1980 (Table I), was plated on glucose minimal medium containing 40 μg/ml histidine and 15 μg/ml adenine as the sole source of purines. Mutants that were still auxotrophic and capable of utilizing adenine as a purine source were characterized further as described under “Results.” Several independent selections were performed. To facilitate subsequent analysis of the mutants obtained, some of these selections were performed on strains that were isogenic with CN1980 except that they contained gsk mutant alleles which did not give rise to kanamycin resistance. Verification of genomic mutations by colony PCR amplification and DNA sequencing was performed as described previously (23Petersen C. Møller L.B. J. Biol. Chem. 2001; 276: 884-894Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). A phage λ-sensitive LamB+ derivative of CN2388–2 (Table I) was mutagenized with mini-Tn10 cam from λNK1324 as described (28Kleckner N. Bender J. Gottesman S. Methods Enzymol. 1991; 204: 139-180Crossref PubMed Scopus (351) Google Scholar). After penicillin enrichment (23Petersen C. Møller L.B. J. Biol. Chem. 2001; 276: 884-894Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), we isolated chloramphenicol-resistant clones that had lost the ability to utilize adenine as a purine source by replica plating onto glucose minimal plates containing either adenine or hypoxanthine as a purine source. One of these mutants, CN2451, gave rise to a PCR product of ∼1.5 kilobases with the cam-down and ade-Bamprimers (Table II), and sequencing of this DNA fragment showed that thecam gene was located at positions 4156–4164 2The GenBank™ accession numbers forade/yicP and hns DNA sequences areAE000444 and AE000222, respectively. within theade gene in the same orientation as ade.Table IIDNA oligonucleotides used for PCR amplifications or primer extension analysesNameSequence (5′–3′)2-aNucleotides complementary to their genome target sequences are shown in capital letters, whereas sequence tags containing restriction sites for subsequent cloning of PCR products are in lowercase letters.#1224CGCCAGGGTTTTCCCAGTCACGACcam-downCAGAGCCTGATAAAAACGGTade-R1ccgaattcaCCATTGGAGGAGATTTAATCCCade-R12ccgaattcAAGCCCGCTTTATCAGTTACACCade-H3ccgaagcttGCTAACAATTCCTGGTATTCAGCCade-BamcgggatccGTGGCATAAACGTAACTGGTGAChns-RVgccgatatCTAAGTCCATGCTCTTATTGCGhns-H3gccaagctTTGCTTGATCAGGAAATCGTCG2-a Nucleotides complementary to their genome target sequences are shown in capital letters, whereas sequence tags containing restriction sites for subsequent cloning of PCR products are in lowercase letters. Open table in a new tab DNA manipulations, transformations, and restriction analyses were performed according to standard procedures (29Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.1982Google Scholar). PCR amplifications were performed on 1 μg of genomic DNA using Pfu polymerase (Stratagene) according to the manufacturer's recommendations. The DNA oligonucleotides used as primers in PCR reactions are described in Table II. The sequence of cloned PCR fragments were verified by sequencing on an ABI377 DNA sequencer (Applied Biosystems/PerkinElmer Life Sciences). For construction of pAde, the ade gene and flanking regions (nucleotides 3612–5598) were PCR-amplified from genomic DNA with theade-R1 and ade-Bam primers (Table II) and inserted between the EcoRI and BamHI sites of the medium-copy vector pBR322 (30Bolivar F. Rodriguez R.L. Greene P.Y. Betlach M.C. Heynecker H.L. Boyer H.W. Crosa Y.H. Falkow S. Gene (Amst.). 1977; 2: 95-113Crossref PubMed Scopus (3526) Google Scholar). For construction of medium-copy ade-lacZ gene fusions, the ade promoter and 65 base pairs of N-terminal coding region was PCR-amplified from genomic DNA and inserted between unique EcoRI and HindIII sites in thelacZ gene fusion vector pCN302, which is based on the pBR322 replicon (24Petersen C. Møller L.B. Gene (Amst.). 2000; 261: 289-298Crossref PubMed Scopus (90) Google Scholar). The ade promoter fragments in pCN2421 and pCN2422 were amplified with the ade-R1 and ade-H3 primers (Table II), whereas the promoter insert in pCN2534 was generated with the ade-R12 and ade-H3 primers (Table II). A low-copy derivative, pCN2515, of the IS1-activatedade-lacZ fusion, pCN2422, was constructed by inserting theade promoter fragment into the low-copy lacZ gene fusion vector, pCN2423, which is based on the pSC101 replicon (23Petersen C. Møller L.B. J. Biol. Chem. 2001; 276: 884-894Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). 30 ml of bacterial culture was harvested on ice atA436 = 0.5. Cells were collected by centrifugation, washed, and resuspended in 50 mm Tris-HCl, pH 7.5, to A436 = 20. Cells were disrupted by sonic treatment, and the extract was cleared by centrifugation at 20,000 × g for 3 min in a refrigerated microcentrifuge. Assays were performed at 37 °C by mixing appropriately diluted extract with [8-14C]adenine (8.4 Ci/mol) at a final concentration of 0.5 mm in a total volume of 50 μl of 40 mm Tris-HCl, pH 7.5, 5 mm MnCl2. At timed intervals, samples of 15 μl were taken out, boiled for 2 min, and cooled on ice. After a 3-min centrifugation at 20,000 × g, 5 μl of supernatant was applied to a polyethyleneimine thin layer chromatography plate. Substrate and products were separated by chromatography in water and subsequently quantitated by counting in an Instant Imager (Packard Instrument Co.). The enzymatic activities were calculated from the initial slope of a plot of the amount of radioactive substrate remaining as a function of time (see Fig.2). The reported activities are the averages of two independent determinations, which deviated less than 10% from the average. The adenine deaminase was strongly dependent on manganese ions; the enzymatic activity decreased more than 30-fold if MnCl2 was omitted from the assay (data not shown). Differential rates of β-galactosidase synthesis were measured at 37 °C as described previously (31Petersen C. Mol. Gen. Genet. 1987; 209: 179-187Crossref PubMed Scopus (48) Google Scholar). Bacterial RNA was prepared by hot phenol extraction, and primer extension analysis was performed on 5 μg of total RNA as described (32Melefors Ö. von Gabain A. Cell. 1988; 52: 893-901Abstract Full Text PDF PubMed Scopus (94) Google Scholar). We used a32P-labeled DNA primer complementary to nucleotides 41–64 of the lacZ coding sequence (#1224, see Table II). Mutants with an activated adenine deaminase gene were sought by plating a purine-requiringdeoD mutant strain, such as CN1980 (Table I), on glucose minimal medium containing adenine and histidine (see “Experimental Procedures”). With a frequency of 10−6, mutant colonies appeared that apparently converted adenine efficiently into guanine nucleotides despite the blocked deoD pathway and the presence of histidine in the medium. Sixty of such independent mutants were characterized further and found to belong to three distinct classes as described in the following. Half of the isolated mutants lost the ability to grow with adenine as a purine source upon introduction of thehisG::Tn10 allele from NK5526 (Table I), and for several of these mutants the responsible mutation was mapped to the hisG gene itself (data not shown). Thus, the feedback regulation of the HisG enzyme was probably eliminated in this class of mutants to allow an increased flux through the histidine biosynthetic pathway even in the presence of histidine. These mutants were not characterized further. The remaining mutants did not lose the ability to grow on adenine upon introduction of a hisG::Tn10 or adeoD::cam mutation (data not shown), suggesting that a novel third pathway for conversion of adenine into guanine nucleotides had been activated. Based on differences with respect to growth and colony morphology, these mutants could be divided into two distinct classes (II and III) consisting of 20 and 10 independent clones, respectively. The Class II mutants, like the parent strain, grew well on a variety of carbon sources and formed normal non-mucoid colonies. Genetic analysis revealed that the responsible mutation in the Class II mutants showed a high cotransduction frequency with the ilvB locus (data not shown), which is compatible with a location in the immediate vicinity of theyicP gene. Thus, we hypothesized that the novel activated pathway in Class II mutants might involve direct deamination of adenine to hypoxanthine by the yicP gene product. In support of this notion, we found that a crude extract of one of the Class II mutants, CN2388–2, efficiently converted adenine into hypoxanthine with no apparent formation of any intermediate products, corresponding to a 20-fold increase of the cellular adenine deaminase activity compared with the parent strain, CN1980 (Fig. 2, left and center panels). Furthermore, we subjected a derivative of CN2388–2 to transposon mutagenesis with mini-Tn10 cam and isolated a clone that had specifically lost the ability to grow with adenine as a purine source while retaining the ability to utilize hypoxanthine. Subsequent analysis revealed that the cam insert in this strain, CN2451, had indeed disrupted the yicP gene (see “Experimental Procedures”). Back transduction of theyicP::cam allele into all the Class II mutants eliminated their ability to use adenine as a purine source (data not shown), confirming that the original phenotype was caused by activation of the yicP gene, which we propose be renamedade. This conclusion was further corroborated by cloning of the chromosomalade gene and its native promoter in a multicopy plasmid, pAde (see “Experimental Procedures”). Introduction of this plasmid increased the adenine deaminase activity of CN1980 ∼30-fold from 0.5 to 15.4 μmol/min/g of dry weight and enabled the transformant to grow well in glucose minimal medium with adenine as the sole source of purines at a rate comparable with that of a purine-prototrophic strain (data not shown). These results indicated that the wild typeade gene encodes a functional adenine deaminase but is simply too poorly expressed at normal gene dosage to satisfy the cellular purine requirement, which is on the order of 4 μmol/min/g of dry weight (33Neidhardt F.C. Ingraham J.L. Schaechter M. Physiology of the Bacterial Cell. Sinauer Associates, Inc., Sunderland, MA1990Google Scholar). Indeed, the low but significant adenine deaminase activity in the wild type strain, CN1980, was completely abolished by the ade::cam disruption in CN2481 (compare Figs. 2, left and right panels). In agreement with the results of Matsui et al. (22Matsui H. Shimaoka M. Kawasaki H. Takenaka Y. Kurahashi O. Biosci. Biotechnol. Biochem. 2001; 65: 1112-1118Crossref PubMed Scopus (24) Google Scholar), theE. coli adenine deaminase was strongly dependent on manganese ions for activity (see “Experimental Procedures”), as also observed for the homologous enzyme from B. subtilis(36Nygaard P. Duckert P. Saxild H.H. J. Bacteriol. 1996; 178: 846-853Crossref PubMed Google Scholar). The mutations responsible for the increased adenine deaminase activity were identified for 15 Class II mutants by PCR amplification and DNA sequencing of the ade promoter region (Fig. 3). In 12 of these mutants theade gene was activated by integration of an IS1 element within the first 100 base pairs of the divergent neighboring gene, yicO. Two mutants were activated by insertion of IS4 or IS5 in the same region of yicO, whereas one mutant contained an IS5 insertion in the intercistronic region between ade and yicO. The latter insertion was the only one found to be located downstream of the ade promoter. In addition we found two Class II mutants with unidentified insertions or rearrangements inyicO that could not be spanned by PCR amplification as well as three mutants that apparently contained an amplification of the entire ade region including the ilvB locus (data not shown). The finding that all the identified Class II mutations mapped outside of the structural ade gene underscored that the cryptic nature of this locus was caused by a low level of expression rather than by malfunctioning of the encoded gene product. It is also noteworthy that activation could occur by insertion of IS elements within an extended region of 160 base pairs surrounding theade promoter and that this activation, at least for IS1, was independent of the orientation of the insertion element. The Class III mutants were clearly distinguishable from the parent strain and the other mutant classes by their formation of mucoid colonies even on LB plates and their poor growth with glycerol or succinate as carbon sources irrespective of the purine source. The cellular adenine deaminase activity of a representative Class III mutant, CN2388-16 (Table I), was found to be 3.8 μmol/min/g of dry weight, which is nearly 8-fold higher than the enzymatic activity of the wild type parent. Furthermore, CN2388-16 lost the ability to grow with adenine as a purine source upon introduction of theade::cam allele. These results indicated that the Class III mutants, like those of Class II, contain an activated ade gene. However, transductional mapping experiments indicated that the responsible activating mutation was unlinked to the ade and ilvB loci (data not shown), suggesting a mutation in a trans-acting regulatory locus. Accordingly, we mapped the mutation responsible for the increased adenine deaminase activity and the other phenotypes of CN2388-16 to the 27-min region of the genome immediately clockwise of thesupF marker (data not shown). This corresponds to the location of the hns gene, mutations in which are known to cause increased formation of capsular polysaccharides and poor growth on gluconeogenic carbon sources (37Sledjeski D. Gottesman S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2003-2007Crossref PubMed Scopus (191) Google Scholar). Thus, we inferred that the Class III mutants might contain loss-of-function mutations in thehns gene, and this was confirmed by PCR amplification of thehns gene in CN2388-16 and another Class III mutant, CN2388–11, using the primers hns-RV + hns-H3 (Table II). DNA sequencing of the PCR fragments revealed that the hns gene in both cases had been disrupted by insertion of an IS1 element in the region encoding the C-terminal DNA binding domain of H-NS,2 leaving a truncated reading frame of 126 or 108 codons, respectively (plus additional codons encoded by the IS1 sequence). In agreement with these results, we found that the cellular adenine deaminase activity increased 11-fold upon introduction of the well characterized hns205::Tn10 allele into CN1980 (Table III). This hnsdisruption allele, which codes for a truncated H-NS protein of only 93 amino acid residues (38Free A. Porter M.E. Deighan P. Dorman C.J. Mol. Microbiol. 2001; 42: 903-918Crossref PubMed Scopus (38) Google Scholar), also mimicked the other phenotypes of the selected Class III mutants with respect to increased mucoidicity and poor growth on glycerol or succinate. All these results indicated that the cryptic nature of the ade gene in wild type strains is due to H-NS-mediated gene silencing.Table IIIEffect of IS insertions and H-NS on the cellular adenine deaminase activity (in μmol/min/g of dry weight)Strain pair (hns+/hns205::Tn10)adealleleAdenine deaminase activityFold repression by H-NShns+hns205::Tn10CN1980/CN2479Wild type0.55.811.0CN2388–2/CN2588yicO-2::IS113.618.91.4CN2388–6/CN2589yicO-6::IS47.213.21.8CN2388–15/CN2590yicO-15::IS15.011.82.4(CN2349/CN2498) + pBR322Wild type0.34.313.8(CN2349/CN2498) + pAdeWild type + pAde6.983.412.0 Open ta" @default.
• W2035940253 created "2016-06-24" @default.
• W2035940253 creator A5000627885 @default.
• W2035940253 creator A5079298381 @default.
• W2035940253 creator A5090647856 @default.
• W2035940253 date "2002-08-01" @default.
• W2035940253 modified "2023-10-03" @default.
• W2035940253 title "The Cryptic Adenine Deaminase Gene of Escherichia coli" @default.
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