We wish to understand how the expression of the Escherichia coli is coordinately regulated during growth rate transitions accompanying physiological and nutritional stress. Interest centers on the pleiotropic regulatory effects mediated by guanosine 3',5'-bispyrophosphate (ppGpp) and focuses on a primary regulatory target inhibited by ppGpp, ribosomal RNa operon transcription. Normally, ppGpp accumulates to high levels that are correlated inversely with nutritional abundance and growth rate. We have devised a means of varying steady state levels of ppGpp independent of nutritional adequacy by isolating mutants in the spoT gene with varying degrees of defects. The spoT gene encodes a ppGpp 3'-pyrophosphatase. Studies with these isongenic strains show that a strong inverse correlation between rRNA P1 promoter activity and ppGpp levels holds over the full physiological range of ppGpp concentration, including those characteristic of normal growth. A similar correlation applies for growth rate itself. The simplicity of this relationship suggests a direct interaction of ppGpp as a negative effector with the P1 promoter during normal growth as well as during the stringent response. This possibility is being tested by the derivation of ppGpp insensitive P1 promoter mutants that have been fused to a galactokinase indicator gene. The spoT gene has been localized within a new operon and is being characterized by chromosomal insertion-deletion analysis. Ribosomal RNa operons display several unusual transcriptional features, including antitermination dual promoters with supercoil-dependent activities, and presumably the ability to stop an antiterminating transcript. We are continuing to dissect out these features and have found a new region of antitermination just upstream of the 23S gene in a position analogous to the known region just upstream of the 16S gene. We have also found antitermination sequences in plasmids are unstable. Wild-type antitermination sequences are considerably more active than spontaneous stable variants.
