Construction and use of pyr::lac fusion strains to study regulation of pyrimidine biosynthesis in Salmonella typhimurium

Michaels, Glenn; Kelln, Rod A.

doi:10.1007/bf00325910

Cited by 9 publications

(10 citation statements)

References 37 publications

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“…Transfer of an out (dctA) mutation to a usp strain was selected by plating on the same basic medium, but with the addition of FOA. Methods for transposon technology with Tn10 were as previously reported (27). Conjugations were carried out as described previously (15).…”

Section: Methodsmentioning

confidence: 99%

“…Accordingly, 5-fluoroorotate (FOA) is a potent growth inhibitor in glycerol minimal medium and has been employed for the selection of pyrH (UMP kinase) mutants of both S. typhimurium (40) and E. coli (15). It has been observed that orotate is growth rate limiting for carAB and pyrBI mutants in a concentration-dependent manner, and culturing cells at varying orotate concentrations has been used as a means to establish partial pyrimidine starvation for purposes of studying the impact of pyrimidine nucleotide limitation on specific gene expression and general aspects of macromolecular synthesis (3,5,27,36,38). In contrast, reducing the concentration of uracil serves to lower only the growth yield, not the growth rate (36), and indicates that the transport of orotate into the cell represents a rate-limiting step.…”

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confidence: 99%

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Utilization of orotate as a pyrimidine source by Salmonella typhimurium and Escherichia coli requires the dicarboxylate transport protein encoded by dctA

et al. 1996

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Mutants deficient in orotate utilization (initially termed out mutants) were isolated by selection for resistance to 5-fluoroorotate (FOA), and the mutations of 12 independently obtained isolates were found to map at 79 to 80 min on the Salmonella typhimurium chromosome. A gene complementing the mutations was cloned and sequenced and found to possess extensive sequence identity to characterized genes for C4-dicarboxylate transport (dctA) in Rhizobium species and to the sequence inferred to be the dctA gene of Escherichia coli. The mutants were unable to utilize succinate, malate, or fumarate as sole carbon source, an expected phenotype of dctA mutants, and introduction of the cloned DNA resulted in restoration of both C4-dicarboxylate and orotate utilization. Further, succinate was found to compete with orotate for entry into the cell. The S. typhimurium dctA gene encodes a highly hydrophobic polypeptide of 45.4 kDa, and the polypeptide was found to be enriched in the membrane fraction of minicells harboring a dctA ؉ plasmid. The DNA immediately upstream of the deduced ؊35 region contains a putative cyclic AMP-cyclic AMP receptor protein complex binding site, thus affording an explanation for the more effective utilization of orotate with glycerol than with glucose as carbon source. The E. coli dctA gene was cloned from a lambda vector and shown to complement C4-dicarboxylate and orotate utilization in FOA-resistant mutants of both E. coli and S. typhimurium. The accumulated results demonstrate that the dctA gene product, in addition to transporting C4-dicarboxylates, mediates the transport of orotate, a cyclic monocarboxylate.In Salmonella typhimurium and Escherichia coli, six enzymic reactions are involved in de novo pyrimidine nucleotide biosynthesis to form UMP, the ordered sequence of the unlinked genes for the enzymes being carAB and pyrBI, -C, -D, -E, and -F. Wild-type cells are not readily permeable to intermediates of the pyrimidine biosynthetic pathway, with the exception of orotate, which is formed from dihydroorotate by the action of dihydroorotate dehydrogenase (pyrD). Orotate satisfies the pyrimidine requirement of carAB, pyrBI, pyrC, or pyrD mutants and is effective at low to moderate concentrations, provided glycerol is used as the carbon source (29,39,40). Accordingly, 5-fluoroorotate (FOA) is a potent growth inhibitor in glycerol minimal medium and has been employed for the selection of pyrH (UMP kinase) mutants of both S. typhimurium (40) and E. coli (15). It has been observed that orotate is growth rate limiting for carAB and pyrBI mutants in a concentration-dependent manner, and culturing cells at varying orotate concentrations has been used as a means to establish partial pyrimidine starvation for purposes of studying the impact of pyrimidine nucleotide limitation on specific gene expression and general aspects of macromolecular synthesis (3,5,27,36,38). In contrast, reducing the concentration of uracil serves to lower only the growth yield, not the growth rate (36), and indicates that the ...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Utilization of orotate as a pyrimidine source by Salmonella typhimurium and Escherichia coli requires the dicarboxylate transport protein encoded by dctA

et al. 1996

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“…The composition of the minimal medium has been reported [14]. The complex media, nutrient broth and L broth, were prepared as described [I 51.…”

Section: Media and Culture Methodsmentioning

confidence: 99%

“…Chromosomal DNA from strain MLX2, a previously characterized pyrB regulatory mutant derived from strain MLXI [14], was digested with EcoRI and cloned into the EcoRI site of pGM5. Ligation of the EcoRI site in lac2 of the donor pyrB: :MudlX DNA in the correct orientation produces a construct with an intact lacZ, the expression of which is governed by the pyrB regulatory region.…”

Section: Pstlmentioning

confidence: 99%

Cloning, nucleotide sequence and expression of the pyr BI operon of Salmonella typhimurium LT2

1987

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The pyrB-pyrI region of the Salmonella typhimurium LT2 chromosome has been cloned and sequenced. The two genes were found to constitute an operon, with pyrl being the distal gene and separated from pyrB by a 15-bp intercistronic region. Sequence analysis revealed the presence of two potential promoters; transcription initiated from the promoter proximal to pyrB would produce a transcript which could direct the synthesis of a 33-aminoacid leader peptide. The leader sequence possesses the requisite features of a rho-independent transcriptional terminator (attenuator) which is positioned 22 bp upstream from the pyrB structural gene. A regulatory mutation imparting a 30-fold elevated expression of pyrBI was identified as a two-base-pair deletion in the track of A . T base pairs located on the 3' side of the region of dyad symmetry of the attenuator. The leader sequence also has an additional region of dyad symmetry (putative transcriptional pause site) located 33 nucleotides upstream from the start of the proposed attenuator. The intervening sequence between the putative pause site and the indicated attenuator is characterized by encoding a high content of uracil residues in the transcript. Construction and analysis of transcriptional and translational fusions provided evidence that (a) the leader region has the necessary features to mediate polypeptide synthesis in vivo, (b) the removal of the region corresponding to the pause site and attenuator results in constitutive expression and (c) the more distant potential promoter does not appear to facilitate significant transcriptional activity. Strong homology exists with the pyrBI operon from E . s c~~r i c~i a coli K-12 but notable differences are observed.The pyrimidine biosynthetic enzyme, aspartate transcarbamoylase from Escherichia coli and Sulnionella typhimurium is composed of six catalytic (c) and six regulatory (r) polypeptides, which are organized as two trimeric catalytic (C) subunits and three dimeric regulatory (R) subunits in the native enzyme (reviewed in [l]). The structural genes for the c and r polypeptides have been designated pyrB and pyrl, respectively, and for E. coli the two genes have been shown to comprise an operon, with pyrB being promoter-proximal Previous studies have shown that expression of pyrBI is regulated over a several-hundredfold range [3 -51 and that regulation of expression occurs at the level of transcription [6]. Sequencing studies on the promoter-regulatory region of pyrBI from E. coli led to the identification of a putative rhoindependent terminator (attenuator) located approximately 20 bp upstream of the pyrB gene [7, 81. Also identified was a transcriptional pause site positioned roughly 20 bp before the start of the attenuator region [8]. The region at the pause site encodes a uridine-rich sequence in the transcript. Transcription initiating from a promoter about 160 bp upstream of pyrB, and proceeding without termination at the attenuator, will produce a transcript which can encode a 44-amino-acid peptide, with the termin...

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“…Hfr derivatives of the pyr-lac+ fusion strains which transfer their chromosome in the direction of pyr gene transcription were constructed by forcing the F'tsll4 lac' zzf-21: :TnlO episome to integrate into the chromosome by lac homology, essentially as described by Michaels and Kelln [27]. Equal volumes of the Salmonella typhimurium strain KP1938 (trp,cys) containing the F'lac' episome and the recipient fusion strains were mixed, incubated at 30 "C for 30min and plated on minimal agar with lactose (0.1%) as carbon source and containing tetracycline (10 pg/ml).…”

Section: Determination Of Direction Of Pyr Gene Transcriptionmentioning

confidence: 99%

Studies on the structure and expression of Escherichia coli pyrC, pyrD and pyrF using the cloned genes

Jensen¹,

Larsen²,

Schack³

et al. 1984

Eur J Biochem

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The Escherichia coli pyrC, pyrD and pyrF genes were cloned on multicopy plasmids derived from pBR322 and analysed by means of restriction endonucleases. It was found that the pyre gene is destroyed by cutting with the restriction endonuclease BamHI, that the entire pyrD gene can be isolated on a 1300‐base pairs DNA fragment generated by EcoRI cleavage and that cutting with EcoRI removes the promotor and probably also the translational start site from the pyrF gene. More details on the restriction maps are presented. Further, it was found that the presence of a pyr gene in multiple copies on a plasmid does not significantly interfere with the activity of the chromosomal pyr genes. Using the ‘minicell’ technique, the polypeptides encoded by the three cloned pyr genes were identified. The relative molecular masses for the pyrC‐encoded and pyrD‐encoded polypeptides are 38000–40000 and 36000–38000, respectively. Thus in their native form, dihydroorotase and dihydroorotate oxidase appear to be dimeric proteins. The ‘minicell’ experiments positively identified a protein chain of Mr 23000–24000 as being a subunit of OMP decarboxylase encoded by pyrF. Moreover, the coding frame for this polypeptide seems to be expressed as the first gene in the operon with the coding frame for another protein chain of Mr 13000–14000. Since, however, the native OMP decarboxylase during sedimentation and gel filtration behaves as a protein of Mr 45000 ± 4000, this latter polypeptide (Mr 13000–14000) is hardly a component of the enzyme. Pyr‐lac + operon fusions were constructed by the Mu d1 procedure. By integrating an F'lac episome into the lac part of the fusions and determining the direction of chromosomal transfer from the resultant Hfr strains, the direction of pyrC transcription was found to be counter‐clockwise, while pyrD and pyrF were found to be transcribed in a clockwise direction.

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Construction and use of pyr::lac fusion strains to study regulation of pyrimidine biosynthesis in Salmonella typhimurium

Cited by 9 publications

References 37 publications

Utilization of orotate as a pyrimidine source by Salmonella typhimurium and Escherichia coli requires the dicarboxylate transport protein encoded by dctA

Utilization of orotate as a pyrimidine source by Salmonella typhimurium and Escherichia coli requires the dicarboxylate transport protein encoded by dctA

Cloning, nucleotide sequence and expression of the pyr BI operon of Salmonella typhimurium LT2

Studies on the structure and expression of Escherichia coli pyrC, pyrD and pyrF using the cloned genes

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