Purine biosynthesis in <i>Escherichia coli</i> K12: structure and DNA sequence studies of the <i>purHD</i> locus

Flannigan, K. A.; Hennigan, Steven H.; Vogelbacker, H.; Gots, Joseph S.; Smith, John L.

doi:10.1111/j.1365-2958.1990.tb00605.x

Cited by 17 publications

(7 citation statements)

References 51 publications

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“…1A). In most bacteria, the genes encoding the proteins required for this pathway are maintained as a 12-gene operon (1,19,20,24). The purH gene is the penultimate gene in the pur operon sequence (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Role of Purine Biosynthesis in Bacillus anthracis Pathogenesis and Virulence

et al. 2011

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Bacillus anthracis, the etiological agent of anthrax, is a spore-forming, Gram-positive bacterium and a category A biothreat agent. Screening of a library of transposon-mutagenized B. anthracis spores identified a mutant displaying an altered phenotype that harbored a mutated gene encoding the purine biosynthetic enzyme PurH. PurH is a bifunctional protein that catalyzes the final steps in the biosynthesis of the purine IMP. We constructed and characterized defined purH mutants of the virulent B. anthracis Ames strain. The virulence of the purH mutants was assessed in guinea pigs, mice, and rabbits. The spores of the purH mutants were as virulent as wild-type spores in mouse intranasal and rabbit subcutaneous infection models but were partially attenuated in a mouse intraperitoneal model. In contrast, the purH mutant spores were highly attenuated in guinea pigs regardless of the administration route. The reduced virulence in guinea pigs was not due solely to a germination defect, since both bacilli and toxins were detected in vivo, suggesting that the significant attenuation was associated with a growth defect in vivo. We hypothesize that an intact purine biosynthetic pathway is required for the virulence of B. anthracis in guinea pigs.

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Section: Resultsmentioning

confidence: 99%

“…The purH gene is the penultimate gene in the pur operon sequence (Fig. 1B), followed by purD (20,24). Therefore, it is possible that a mutation in the purH gene results in a polar effect on purD.…”

Section: Resultsmentioning

confidence: 99%

Role of Purine Biosynthesis in Bacillus anthracis Pathogenesis and Virulence

et al. 2011

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“…After recovery, the DNA fragment was treated with T4 DNA polymerase to create blunt ends (Flannigan et al, 1990), digested with Pstl, and cloned into the EcoKW-Pstl sites of the Bluescript vector, Ml3 KS+ (Stratagene, Inc., San Diego, CA). The resulting ligation mix was used to transform strain XLl-Blue with selection for Amp on LB-agar plates supplemented with ampicillin (100 jtg/mL), X-Gal, and IPTG (Messing, 1983).…”

Section: Methodsmentioning

confidence: 99%

“…In order to recognize potential transcription errors by Taq polymerase, three independent clones were identified and retained for DNA sequencing. The sequences were determined as previously described (Flannigan et al, 1990;Cheng et al, 1990). After confirmation of the DNA sequence, the modified purC gene was then transferred into the XpL expression vector, pJS338, via the Bamlll and Pstl restriction sites and transformed into the host strain TX635 as described above for the purEK expression vector.…”

Section: Methodsmentioning

confidence: 99%

Purification and characterization of the purE, purK, and purC gene products: identification of a previously unrecognized energy requirement in the purine biosynthetic pathway

Meyer¹,

Leonard²,

Bhat³

et al. 1992

Biochemistry

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159

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Aminoimidazole riobnucleotide carboxylase, the sixth step in the purine biosynthetic pathway, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to carboxyaminoimidazole ribonucleotide (CAIR). The gene products of the purE and purK genes (PurE and PurK, respectively) thought to be responsible for this activity have been overexpressed and the proteins purified to homogeneity. PurE separates from PurK in the first ammonium sulfate fractionation during the purification. No evidence for association of the two gene products under a variety of conditions using a variety of methods could be obtained. To facilitate the assay for CAIR production, the purC gene product, 5-aminoimidazole-4-N-succinylcarboxamide ribonucleotide (SAICAR) synthetase has also been overexpressed and purified to homogeneity. The activities of PurE, PurK, and PurE.PurK have been investigated. PurE alone is capable of catalyzing the conversion of AIR to CAIR 1 million times faster than the nonenzymatic rate. The Km for HCO3- in the PurE-dependent reaction is 110 mM! PurK possesses an ATPase activity that is dependent on the presence of AIR. No bicarbonate dependence on this reaction could be demonstrated (less than 100 microM), and AIR is not carboxylated during the hydrolysis of ATP. Incubation of a 1:1 mixture of PurE and PurK at low concentrations of bicarbonate (less than 100 microM) revealed that CAIR is produced but requires the stoichiometric conversion of ATP to ADP and Pi. No dependence on the concentration of HCO3- could be demonstrated. A new energy requirement in the purine biosynthetic pathway has been established.

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“…This site contains a version of the dyad symmetry ACGCAAACGTTTGCGT (39,40), orig-inally predicted to be the site of purR regulatory protein binding on the basis of comparisons of the control regions of the different pur loci (16,26,44,47). The purC locus is subject to regulation by the purR gene product (1, 11,16).…”

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DNA sequence of the purC gene encoding 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase and organization of the dapA-purC region of Escherichia coli K-12

et al. 1990

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5'-Phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase (EC 6.3.2.6), encoded by the purC gene of Escherichia coli K-12, catalyzes the synthesis of 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide from 5'-phosphoribosyl-5-aminoimidazole-4-carboxylic acid. The mature protein, as deduced from the purC structural gene sequence, contains 237 amino acids and has a calculated Mr of 26,998. The control region of the purC gene was identified by primer extension mapping of the 5' end of the purC mRNA. The purC control region contains a binding site for and is regulated by the purine repressor, the product of the purR gene. An unusual feature of the 5' untranslated region of the purC mRNA is the presence of a repetitive extragenic palindrome sequence normally found in intercistronic or 3' untranslated regions. The DNA sequence was extended 1.281 kilobases upstream of the purC structural gene and overlapped with the previously determined dapA sequence. Termination of transcription from the dapA-purC intercistronic region may occur within the -35 region of the purC control region. The purC gene has been positioned on the E. coli restriction map and is transcribed in a counterclockwise direction.

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Purine biosynthesis in Escherichia coli K12: structure and DNA sequence studies of the purHD locus

Cited by 17 publications

References 51 publications

Role of Purine Biosynthesis in Bacillus anthracis Pathogenesis and Virulence

Role of Purine Biosynthesis in Bacillus anthracis Pathogenesis and Virulence

Purification and characterization of the purE, purK, and purC gene products: identification of a previously unrecognized energy requirement in the purine biosynthetic pathway

DNA sequence of the purC gene encoding 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase and organization of the dapA-purC region of Escherichia coli K-12

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