Abstract
Escherichia coli has two enzymes catalyzing the synthesis of guanosine tetraphosphate (ppGpp), designated ppGpp synthetase I (PSI = RelA) and II (PSII), whose activities are regulated differently. Until now, the gene for PSII had not been identified. Here, an E. coli relA1 strain that expresses lacZ from an rrnB P1 promoter was used to screen mutants with increased beta-galactosidase activity on 5-bromo-4-chloro-3-indoyl beta-D-galactoside indicator plates at 30 degrees C. About 15% of the mutants obtained in this manner had reduced levels of ppGpp at 30 degrees C and no detectable ppGpp at 43 degrees C. These mutants did not form colonies at 42 degrees C on minimal medium plates and had elevated ribosome concentrations and higher growth rates at 30 degrees C. Genetic mapping by phage P1 transduction and complementation analyses showed that the mutations were located in spoT and that they were recessive. Specific inhibition of SpoT-dependent ppGpp degradation activity with picolinic acid showed that two of the mutants tested were deficient in ppGpp synthesis activity. These results indicate that spoT is required for PSII activity, suggesting that spoT encodes both ppGpp degradation and synthesis activities and that these two functions can be affected independently by mutation.
Highlights
The activities of the two ppGpp-synthesizing enzymes, PSI and PSII, are regulated differently in response to environmental stimuli
The parental strainVH273 wasconstructed with antibiotic resistance markers kan and tet near P1-lacZ and SPOTr,espectively, t o allow screening for such mutations by P1 phage transduction
Following inhibition of SpoT activity by addition of picolinic acidto a final concentrationof 1.5 mM, the parent strain VH273 accumulated 4-fold higher ppGpp levels than mutations isolated inactivate a repressor of spoT; such mu- VH600 and VH730, chosen as representative mutant strains, tations would reduce ppGpp accumulation andwould be within 1 h (Fig. 2 A )
Summary
Rationale for PSZZ Mutant Isolation-The strain VH273 (relAl Alac-argF;Table I) carries an rrnBP1 promoter fused to lacZ and recombined into the chromosome at a location (91.5 min) and in an orientation simulating the normal rrnB locus. @-Gal synthesisin this strain is strongly inhibited by ppGpp (Hernandezand Bremer, 1990). A weak ribosome binding site from the bacteriophage @X174E gene serves as the lacZ translation start toreduce expression from the strong rrn P1 promoter, which allowed the distinction of LacZ-up mutations on X-Gal indicator plates Such mutants with increased expression from the rrnP1 promoter were expected to include mutants with decreased levels of ppGpp due to decreased PSII activity. Nine more mutants which had the properties of increased rrn P1-lacZ expression, lowered ppGpp accumulation, thermosensitivity, and linkage of these phenotypes to spoT were identified, corresponding to a totalof 13 outof the original 89 isolates. Backcrossing these mutationsinto unmutagenized strains carrying the rrn P1-lacZ reporter cassette (see "Experimental Procedures") was sufficient to confer the mutantphenotypes of the original isolates
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