title: Suppressors of dGTP Starvation in Escherichia coli.
abstract: dGTP starvation, a newly discovered phenomenon in whichEscherichia colicells are starved specifically for the DNA precursor dGTP, leads to impaired growth and, ultimately, cell death. Phenomenologically, it represents an example of nutritionally induced unbalanced growth: cell mass amplifies normally as dictated by the nutritional status of the medium, but DNA content growth is specifically impaired. The other known example of such a condition, thymineless death (TLD), involves starvation for the DNA precursor dTTP, which has been found to have important chemotherapeutic applications. Experimentally, dGTP starvation is induced by depriving anE. coligpt optA1strain of its required purine source, hypoxanthine. In our studies of this phenomenon, we noted the emergence of a relatively high frequency of suppressor mutants that proved resistant to the treatment. To study such suppressors, we used next-generation sequencing on a collection of independently obtained mutants. A significant fraction was found to carry a defect in the PurR transcriptional repressor, controllingde novopurine biosynthesis, or in its downstreampurEKoperon. Thus, upregulation ofde novopurine biosynthesis appears to be a major mode of overcoming the lethal effects of dGTP starvation. In addition, another large fraction of the suppressors contained a large tandem duplication of a 250- to 300-kb genomic region that included thepurEKoperon as well as theacrAB-encoded multidrug efflux system. Thus, the suppressive effects of the duplications could potentially involve beneficial effects of a number of genes/operons within the amplified regions.IMPORTANCEConcentrations of the four precursors for DNA synthesis (2'-deoxynucleoside-5'-triphosphates [dNTPs]) are critical for both the speed of DNA replication and its accuracy. Previously, we investigated consequences of dGTP starvation, where the DNA precursor dGTP was specifically reduced to a low level. Under this condition,E. colicells continued cell growth but eventually developed a DNA replication defect, leading to cell death due to formation of unresolvable DNA structures. Nevertheless, dGTP-starved cultures eventually resumed growth due to the appearance of resistant mutants. Here, we used whole-genome DNA sequencing to identify the responsible suppressor mutations. We show that the majority of suppressors can circumvent death by upregulating purinede novobiosynthesis, leading to restoration of dGTP to acceptable levels.
authors.affiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Itsko
authors.affiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA schaaper@niehs.nih.gov.
authors.forename: Roel M
authors.initials: RM
authors.lastname: Schaaper
mesh_terms: D053898:Biosynthetic Pathways / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D004252:DNA Mutational Analysis; D003848:Deoxyguanine Nucleotides / Q000172:deficiency* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D020440:Gene Duplication; D059014:High-Throughput Nucleotide Sequencing; D011687:Purines / Q000096:biosynthesis; D012097:Repressor Proteins / Q000235:genetics; D013489:Suppression, Genetic
chemical_list: C000596829:AcrAB-TolC protein, E coli; D002352:Carrier Proteins; D003848:Deoxyguanine Nucleotides; D029968:Escherichia coli Proteins; C489317:PurR protein, E coli; D011687:Purines; D012097:Repressor Proteins; C029603:deoxyguanosine triphosphate; C030985:purine
keywords: dGTP starvation; purine metabolism; suppressors; tandem duplications; whole-genome sequencing