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A radioimmunoassay for (p)ppGpp and its application to Streptomyces hygroscopicus
Abstract:
Aradioimmunoassay for (p)ppGpp was set up and applied to Streptomyces hygroscopicus. The (p)ppGpp specific antibodies obtained from rabbits immunized with ppGpp-human serum albumin could not distinguish between guanosine-5'-triphosphate-3'-diphosphate (pppGpp) and guanosine-5'-diphosphate-3'-diphosphate (ppGpp) whereas the cross reacitivity against structurally related nucleotides was negligible. Therefore, the antibodies were used for determination of (p)ppGpp, i.e. the sum of pppGpp and ppGpp. The pretreatment of biological samples for quantification of (p)ppGpp included formic acid extraction of cell pool followed by step gradient elution on DEAE-Sephadex A25 (Cl-) column for separation of (p)ppGpp from nucleotides with three or less phosphate groups. The very sensitive radioimmunoassay allowed determination of very low amounts: 1--50 pmoles (p)ppGpp per assay tube. The application of the RIA to S. hygroscopicus revealed a basal level in exponentially growing mycelium of about 2 pmoles (p)ppGpp per mg dry weight. The equilibrated pool size after induction of stringent response with serine hydroxamate was found to be about 30 pmoles (p)ppGpp per mg dry weight.
Insights
A new radioimmunoassay accurately measures guanosine-5'-triphosphate-3'-diphosphate (pppGpp) and guanosine-5'-diphosphate-3'-diphosphate (ppGpp) in Streptomyces hygroscopicus. This method reveals significant changes in (p)ppGpp levels during the stringent response.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- The stringent response is a crucial survival mechanism in bacteria.
- Quantifying guanosine polyphosphates, such as pppGpp and ppGpp, is essential for understanding this response.
- Existing methods for (p)ppGpp quantification have limitations.
Purpose of the Study:
- To develop and validate a sensitive radioimmunoassay (RIA) for quantifying (p)ppGpp in Streptomyces hygroscopicus.
- To apply the developed RIA to measure (p)ppGpp levels during normal growth and under stringent conditions.
Main Methods:
- Development of a radioimmunoassay using antibodies specific to ppGpp.
- Pretreatment of biological samples involving formic acid extraction and DEAE-Sephadex A25 chromatography.
- Quantification of (p)ppGpp (sum of pppGpp and ppGpp) using the developed RIA.
Main Results:
- The RIA demonstrated high specificity for (p)ppGpp with negligible cross-reactivity to other nucleotides.
- The assay is highly sensitive, capable of detecting 1-50 pmoles of (p)ppGpp.
- Basal (p)ppGpp levels in exponentially growing S. hygroscopicus were approximately 2 pmoles/mg dry weight.
- Upon induction of the stringent response with serine hydroxamate, (p)ppGpp levels increased to about 30 pmoles/mg dry weight.
Conclusions:
- A robust and sensitive RIA for (p)ppGpp has been established and validated.
- The RIA provides a valuable tool for studying bacterial stress responses and nucleotide metabolism.
- Significant accumulation of (p)ppGpp occurs during the stringent response in S. hygroscopicus, highlighting its regulatory role.
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