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Occurrence of leu+ revertants under starvation cultures in Escherichia coli is growth-dependent.

ABSTRACT: BACKGROUND:Many investigations have reported that advantageous mutations occurred more frequently under selective conditions than those under non-selective conditions. This phenomenon is referred to as adaptive mutation. Their characteristics are that adaptive mutations are directed and growth-independent. The idea of directed adaptive mutation had been objected by some reports, however, the idea of growth-independent adaptive mutation has been held till today. RESULTS:In this paper, we have observed that under leucine starvation conditions, leu+ revertants accumulated as a function of time; leu- to leu+ reverse mutation rates and frequencies were higher than those under non starvation conditions; and no divided cells could be monitored by the penicillin method. These results were similar to the time-dependent manner of adaptive mutation from previous reports. However, leucine concentration determinate experiments revealed that certain traces of leucine, which leaked from the E. coli cells, was almost always present in the culture. More numbers of leu+ revertants appeared when the similar cultures were dropped in small areas on the selective plates than when spread on the whole selective plates. These results have shown that mutations under leucine starving conditions are growth-dependent. Fluctuation analysis of leu+ revertants indicated that leu-leu+ mutation occurred spontaneously and randomly. In addition, the spectra of leuB gene in the revertants proved that mutations under selective conditions were not specific or directed. CONCLUSIONS:The above investigations led to the conclusion (1) that the occurrence of leu+ mutations under starvation conditions was growth-dependent. The occurrence mutations was also similar to that under non-starvation conditions (2). Under starvation conditions the mutation rates were higher, and was not constant during the long process.

SUBMITTER: Jin J

PROVIDER: S-EPMC115868 | biostudies-literature | 2002 Apr

REPOSITORIES: biostudies-literature

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Occurrence of leu+ revertants under starvation cultures in Escherichia coli is growth-dependent.

Jin Jianling J Gao Peiji P Mao Yumin Y

BMC genetics 20020425

<h4>Background</h4>Many investigations have reported that advantageous mutations occurred more frequently under selective conditions than those under non-selective conditions. This phenomenon is referred to as adaptive mutation. Their characteristics are that adaptive mutations are directed and growth-independent. The idea of directed adaptive mutation had been objected by some reports, however, the idea of growth-independent adaptive mutation has been held till today.<h4>Results</h4>In this pap ...[more]

PMID: 12019019

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Project description:dGTP starvation, a newly discovered phenomenon in which Escherichia coli cells 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 an E. coligpt optA1 strain 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, controlling de novo purine biosynthesis, or in its downstream purEK operon. Thus, upregulation of de novo purine 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 the purEK operon as well as the acrAB-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.IMPORTANCE Concentrations 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. coli cells 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 purine de novo biosynthesis, leading to restoration of dGTP to acceptable levels.

Project description:UnlabelledOur laboratory recently discovered that Escherichia coli cells starved for the DNA precursor dGTP are killed efficiently (dGTP starvation) in a manner similar to that described for thymineless death (TLD). Conditions for specific dGTP starvation can be achieved by depriving an E. coli optA1 gpt strain of the purine nucleotide precursor hypoxanthine (Hx). To gain insight into the mechanisms underlying dGTP starvation, we conducted genome-wide gene expression analyses of actively growing optA1 gpt cells subjected to hypoxanthine deprivation for increasing periods. The data show that upon Hx withdrawal, the optA1 gpt strain displays a diminished ability to derepress the de novo purine biosynthesis genes, likely due to internal guanine accumulation. The impairment in fully inducing the purR regulon may be a contributing factor to the lethality of dGTP starvation. At later time points, and coinciding with cell lethality, strong induction of the SOS response was observed, supporting the concept of replication stress as a final cause of death. No evidence was observed in the starved cells for the participation of other stress responses, including the rpoS-mediated global stress response, reinforcing the lack of feedback of replication stress to the global metabolism of the cell. The genome-wide expression data also provide direct evidence for increased genome complexity during dGTP starvation, as a markedly increased gradient was observed for expression of genes located near the replication origin relative to those located toward the replication terminus.ImportanceControl of the supply of the building blocks (deoxynucleoside triphosphates [dNTPs]) for DNA replication is important for ensuring genome integrity and cell viability. When cells are starved specifically for one of the four dNTPs, dGTP, the process of DNA replication is disturbed in a manner that can lead to eventual death. In the present study, we investigated the transcriptional changes in the bacterium E. coli during dGTP starvation. The results show increasing DNA replication stress with an increased time of starvation, as evidenced by induction of the bacterial SOS system, as well as a notable lack of induction of other stress responses that could have saved the cells from cell death by slowing down cell growth.

Project description:To cope with fluctuations in their environment, bacteria have evolved multiple adaptive stress responses. One such response is the nitrogen regulation stress response, which allows bacteria, such as Escherichia coli, to cope with and overcome conditions of nitrogen limitation. This response is directed by the two-component system NtrBC, where NtrC acts as the major transcriptional regulator to activate the expression of genes to mount the response. Recently, my colleagues and I showed that NtrC directly regulates the expression of the relA gene, the major (p)ppGpp synthetase in E. coli, coupling the nitrogen regulation stress and stringent responses. As elevated levels of (p)ppGpp have been implicated in the formation of persister cells, here, I investigated whether nitrogen starvation promotes their formation and whether the NtrC-RelA regulatory cascade plays a role. The results reveal that nitrogen-starved E. coli synthesizes (p)ppGpp and forms a higher percentage of persister cells than nonstarved cells and that both NtrC and RelA are important for these processes. This study provides novel insights into how the formation of persisters can be promoted in response to a nutritional stress.IMPORTANCE Bacteria often reside in environments where nutrient availability is scarce; therefore, they have evolved adaptive responses to rapidly cope with conditions of feast and famine. Understanding the mechanisms that underpin the regulation of how bacteria cope with this stress is a fundamentally important question in the wider context of understanding the biology of the bacterial cell and bacterial pathogenesis. Two major adaptive mechanisms to cope with starvation are the nitrogen regulation (ntr) stress and stringent responses. Here, I describe how these bacterial stress responses are coordinated under conditions of nitrogen starvation to promote the formation of antibiotic-tolerant persister cells by elevating levels of the secondary messenger (p)ppGpp.

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Occurrence of leu+ revertants under starvation cultures in Escherichia coli is growth-dependent.

Publications

Occurrence of leu+ revertants under starvation cultures in Escherichia coli is growth-dependent.

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