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Involvement of CysB and Cbl regulatory proteins in expression of the tauABCD operon and other sulfate starvation-inducible genes in Escherichia coli.

ABSTRACT: Starvation for sulfate results in increased synthesis of several proteins in Escherichia coli. Among these Ssi (sulfate starvation-induced) proteins are the products of the tauABCD genes, which are required for utilization of taurine as sulfur source for growth. In this study, the role of the cbl gene in expression of tauABCD and other ssi genes was investigated. The protein encoded by cbl shows high sequence similarity to CysB, the LysR-type transcriptional activator of the genes involved in cysteine biosynthesis. Strain EC2541, which contains an internal deletion in cbl, was unable to utilize taurine and other aliphatic sulfonates as sulfur sources. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that many of the Ssi proteins were not synthesized in EC2541. Expression of a translational tauD'-'lacZ fusion required the presence of both cbl and cysB. The interactions of CysB and Cbl with the promoter region of tauABCD were studied by using gel mobility shift experiments and DNase I footprinting. CysB occupied multiple binding sites, whereas Cbl occupied only one site from 112 to 68 bp upstream of the transcription start site. Acetylserine, the inducer of transcription of CysB-regulated genes, stimulated binding of CysB but not of Cbl. Sulfate had no effect on binding of both proteins to the tauABCD promoter region. These results indicate that Cbl is a transcription factor for genes required for sulfonate-sulfur utilization and maybe for other genes whose expression is induced by sulfate starvation.

SUBMITTER: van der Ploeg JR

PROVIDER: S-EPMC179728 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

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Involvement of CysB and Cbl regulatory proteins in expression of the tauABCD operon and other sulfate starvation-inducible genes in Escherichia coli.

van der Ploeg J R JR Iwanicka-Nowicka R R Kertesz M A MA Leisinger T T Hryniewicz M M MM

Journal of bacteriology 19971201 24

Starvation for sulfate results in increased synthesis of several proteins in Escherichia coli. Among these Ssi (sulfate starvation-induced) proteins are the products of the tauABCD genes, which are required for utilization of taurine as sulfur source for growth. In this study, the role of the cbl gene in expression of tauABCD and other ssi genes was investigated. The protein encoded by cbl shows high sequence similarity to CysB, the LysR-type transcriptional activator of the genes involved in cy ...[more]

PMID: 9401024

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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.

Dataset Information

Involvement of CysB and Cbl regulatory proteins in expression of the tauABCD operon and other sulfate starvation-inducible genes in Escherichia coli.

Publications

Involvement of CysB and Cbl regulatory proteins in expression of the tauABCD operon and other sulfate starvation-inducible genes in Escherichia coli.

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