The spoT gene of Escherichia coli encodes a
guanosine 3',5'-bis(diphosphate) 3'-pyrophosphohydrolase (
ppGppase) as well as an apparent
guanosine 3',5'-bis(diphosphate)
synthetase (designated PSII). To determine the regions of the SpoT
protein that are required for these two competing activities, we analysed plasmid-borne deletion mutations for their ability to
complement chromosomal mutations defective in each activity. We found that a region containing the first 203
amino acids of the 702-amino-acid SpoT
protein was sufficient for
ppGppase activity while an overlapping region containing residues 67-374 was sufficient for PSII activity. These data indicate that the catalytic sites involved in the two activities are separate but closely linked in the primary sequence of the SpoT
protein. A
ppGppase-defective delta 1-58 deletion mutant strain failed to synthesize
ppGpp in response to nutrient limitation, also supporting the notion that PSII activity from wild-type SpoT does not increase in response to nutrient limitation. Using a strain lacking PSII activity but retaining
ppGppase activity, we determined the contribution of the RelA
protein (
ppGpp synthetase I, PSI) to
ppGpp synthesis following
glucose starvation. We found that the RelA
protein activity accounts for the initial burst of
ppGpp synthesis at the onset of
glucose starvation but that this source of synthesis is absent when
amino acids are present during
glucose starvation.