Abstract
Intracellular levels of guanosine 3',5'-bispyrophosphate (ppGpp) governed by the relA gene are normally regulated by aminoacyl-tRNA availability for protein synthesis. An experimental system is described in which cellular levels of ppGpp are controlled instead by induction of plasmid pKK223-3 derivatives with the relA structural gene, or portions thereof, under control of the Ptac promoter. In amino acid-rich media, isopropyl-1-thio-beta-D-galactopyranoside induction of transcription of the wild type relA gene in pSM10 yields about a 100-fold overexpression of a metabolically stable, full length (743 amino acid) RelA protein to levels approximating the number of cellular ribosomes. This overexpression is accompanied by a roughly parallel and relC-dependent elevation of ppGpp levels. Induction of a relA gene deletion mutant in pSM11 containing 455 amino-terminal amino acids results in much lower levels of expression of a metabolically unstable 55-kDa protein and elevated ppGpp levels that are almost equivalent to induced pSM10 and are relC-independent. Induction of a larger deletion in pSM12 containing 331 amino-terminal amino acids does not provoke ppGpp accumulation. We are able to elicit high levels of ppGpp without changing nutritional abundance and without massive overexpression of the RelA protein by inducing the metabolically unstable, truncated RelA protein. We find the effects of elevated ppGpp levels to include a slowing of growth, an inhibition of stable RNA accumulation, an inhibition of cellular rrn P1 promoter activities as measured by primer extension, and changes in the pattern of gene expression viewed by two-dimensional electrophoresis of cellular proteins.
Highlights
The nucleotide guanosine 3’,5‘-bispyrophosphate is a candidate for a putative signal molecule whose intracellular concentration can be inversely correlated with growth rate and the rate of ribosomal RNA transcription [5,6,7,8,9] and directly correlated with the expression of certain amino acid operons [10, 11].This nucleotide accumulates to high levels upon amino acid starvation in wild type cells but not in cells containing a relA deletion or a relAl mutation [12]
Strain JM109 is listed as relA2 [17], our experience is that it responds to serine hydroxamate by accumulating ppGpp much the same as a classically wild type relA+strain and immunoblots with anti-RelA antibody detect the characteristic 84-kDa wild type RelA protein
Cells were grown at 37 “Cin MOPS glucose medium [19] containing 0.4 mM phosphate as well as all amino acids at concentrations described by Davis et al [20] except for glutamate and glutamine; methionine was omitted when cellswere labeledwith [35S]methionine
Summary
The focus of this work is to exploit these constructions as ameans of inducing high intracellular concentrations of ppGpp under conditions of constant nutritional abundance in which cellular physiology is minimally perturbed In thecourse of these studies, a relA deletion is characterized in which ppGpp synthetic activity is found to be uniquely relC-independent. The pSM12 plasmid has relA sequences extending from the same ClaI site as pSMlO and pSM11, but thecarboxyl terminus of the gene was further deleted by digestion of the PstI-cut fragment with T4 DNA polymerase [22]to nucleotide position 1272in the relA sequence [16] This protein contains 331 amino acids from the relA gene and, when expressed, migrates as a 37-kDa protein (see “Results”). After incubation in ice for 30 min, the samples were filtered on glass fiber filters (Gelman FGC), washed with trichloroacetic acid, washed with ethanol, and counted by liquid scintillation counting
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