Guanosine tetraphosphate (ppGpp) dependence of the growth rate control of rrnB P1 promoter activity in Escherichia coli.
We have fused the ribosomal RNA promoter P1 from the rrnB gene of Escherichia coli to lacZ and examined its guanosine tetraphosphate (ppGpp)-dependent expression at different growth rates. The rrnB P1-lacZ fusion was constructed on plasmid vectors and then recombined into the chromosome of a delta lac relA1 strain close to the normal location of the rrnB locus and in the normal orientation, coincident with the direction of replication. A series of spoT strains differing in the severity of their SpoT defect were analyzed with respect to their growth characteristics and ppGpp metabolism. The spoT203 allele was introduced into the rrnB P1-lacZ fusion containing strain and used to manipulate the ppGpp concentration independently of the growth medium. 1) The levels of ppGpp during exponential growth are decreased in rich media due to a decreased activity of (p)ppGpp synthetase II (PSII). 2) The specific activity of rrn P1-directed beta-galactosidase was increased in a parabolic fashion with increasing growth rate. A theoretical analysis showed that this was to be expected since enzyme expression from a stringently controlled promoter is affected by changes in the growth rate both via the control of the promoter, and indirectly via the control of bulk mRNA synthesis. 3) The observer specific enzyme activities were converted into rrnB P1 promoter activities, given as lacZ transcription relative to the total rate of transcription. The rrn P1 promoter activity decreased exponentially with increasing cytoplasmic concentration of ppGpp, independent of whether a given level of ppGpp was achieved by using different growth media or by using a spoT allele. These results support two hypotheses: (i) that RNA polymerase is partitioned by ppGpp into two fractions with different abilities to initiate transcription at rrn P1 promoters; and (ii) that during exponential growth ppGpp is derived from ppGpp synthetase II (PSII). Together these hypotheses predict that the growth rate control of rRNA synthesis reflects a control of PSII activity which is regulated by the composition of the growth medium.
Hernandez
V J
VJ
Molecular & Cell Biology Programs, University of Texas, Dallas, Richardson 75083-0688.
Bremer
H
H
D002701:Chloramphenicol / Q000494:pharmacology; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D005798:Genes, Bacterial; D005838:Genotype; D006150:Guanine Nucleotides / Q000378:metabolism*; D006159:Guanosine Tetraphosphate / Q000378:metabolism*; D007700:Kinetics; D008962:Models, Theoretical; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012335:RNA, Ribosomal / Q000235:genetics*; D015183:Restriction Mapping; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D003470:Culture Media; D006150:Guanine Nucleotides; D012335:RNA, Ribosomal; D006159:Guanosine Tetraphosphate; D002701:Chloramphenicol; D001616:beta-Galactosidase