Activities of constitutive promoters in Escherichia coli

The in vivo activities of seven constitutive promoters in Escherichia coli have been determined as functions of growth rate in wild-type relA(+) spoT(+) strains with normal levels of guanosine tetraphosphate (ppGpp) and in ppGpp-deficient Delta relA Delta spoT derivatives. The promoters include (i) the spc ribosomal protein operon promotor P-spc; (ii) the beta-lactamase gene promotor P-bla of plasmid pBR322; (iii) the P-L promoter of phage lambda; (iv) and (v) the replication control promoters P-RNAI and P-RNAII of plasmid pBR322; and (vi) and (vii) the P1 and P2 promoters of the rrnB ribosomal RNA operon. Each strain carried an operon fusion consisting of one of the respective promoter regions linked to lacZ and recombined into the chromosome at the mal locus of a lac deletion strain. The amount of 5'-terminal lacZ mRNA and of beta-galactosidase activity expressed from these promoters were determined by standard hybridization or enzyme activity assays, respectively. In addition, DNA, RNA and protein measurements were used to obtain information about gene dosage, rRNA synthesis and translation rates. By combining lacZ mRNA hybridization data with gene dosage and rRNA synthesis data, the absolute activity of the different promoters, in transcripts/minute per promoter, was determined. In ppGpp-proficient (relA(+) spoT(+)) strains, the respective activities of rrnB P1 and P2 increased 40 and fivefold with increasing growth rate between 0.7 and 3.0 doublings/hour. The activities of P-spc, P-L, P-bla, and P-RNAI increased two- to threefold and reached a maximum at growth rates above 2.0 doublings/hour. In contrast, P-RNAII activity decreased threefold over this range of growth rates. In ppGpp-deficient (Delta relA Delta spoT) bacterial strains, the activities of rrnB P1 and P2 promoters both increased about twofold between 1.6 and 3.0 doublings/hour, whereas the activities of P-spc, P-L, P-bla, and P-RNAI, and P-RNAII were about constant. To explain these observations, we suggest that the cellular concentration of free RNA polymerase increases with increasing growth rate; for saturation the P1 and P2 rRNA promoters require a high RNA polymerase concentration that is approached only at the highest growth rates, whereas the other promoters are saturated at lower polymerase concentrations achieved at intermediate growth rates. In addition, the data indicate that the respective rrnB P1 and P-RNAII promoters were under negative and positive control by ppGpp. This caused a reduced activity of rrnB P1 and an increased activity of P-RNAII during slow growth in wild-type (relA(+) spoT(+)) relative to ppGpp-deficient (Delta relA Delta spoT) bacterial strains. (C) 1999 Academic Press.