Stringent and growth control of rRNA synthesis in Escherichia coli are both mediated by ppGpp

Weak stringent or relaxed responses were induced in Escherichia coli (relA+), using mild amino acid starvation or treatment with chloramphenicol at low concentrations, respectively, such that the growth rate was barely reduced. In this manner, the intracellular concentration of the nucleotide guanosine tetraphosphate, ppGpp, could be varied in any desired range between 0 and 1000 pmol of ppGpp per OD460 unit of culture mass. At the same time, the rate of synthesis of stable RNA (rs; rRNA and tRNA) was measured, relative to the total instantaneous rate of RNA synthesis (rt). The correlation between the cytoplasmic concentration of ppGpp and stable RNA gene activity (rs/rt) was the same as that observed previously with relA+ and relA strains growing exponentially at different rates in different media. This suggests that the distinction between growth control and stringent control of stable RNA synthesis is arbitrary, and that both kinds of control reflect the same ppGpp-dependent phenomenon. By increasing the stable RNA gene dosage, using high copy number plasmids carrying an rrn gene, we have tested the idea that ppGpp partitions the bacterial RNA polymerase into two forms with different probabilities to initiate at stable RNA and mRNA promoters. The relaxed response was not significantly altered, but the extent of the stringent response was reduced by the presence of extra rrn genes. The results agree with quantitative predictions derived from the RNA polymerase partitioning hypothesis.     

Regulation of the Escherichia coli rrnB P2 promoter

The seven rRNA operons in Escherichia coli each contain two promoters, rrn P1 and rrn P2. Most previous studies have focused on the rrn P1 promoters. Here we report a systematic analysis of the activity and regulation of the rrnB P2 promoter in order to define the intrinsic properties of rrn P2 promoters and to understand better their contributions to rRNA synthesis when they are in their natural setting downstream of rrn P1 promoters. In contrast to the conclusions reached in some previous studies, we find that rrnB P2 is regulated: it displays clear responses to amino acid availability (stringent control), rRNA gene dose (feedback control), and changes in growth rate (growth rate-dependent control). Stringent control of rrnB P2 requires the alarmone ppGpp, but growth rate-dependent control of rrnB P2 does not require ppGpp. The rrnB P2 core promoter sequence (-37 to +7) is sufficient to serve as the target for growth rate-dependent regulation.     

Control of rRNA synthesis in Escherichia coli at increased rrn gene dosage. Role of guanosine tetraphosphate and ribosome feedback.

The effects of extra, plasmid-borne rRNA genes on the synthesis rate of rRNA in Escherichia coli were examined by measuring the fraction of total RNA synthesis that is rRNA and tRNA (rs/rt), the cytoplasmic concentration of guanosine tetraphosphate (ppGpp), and the absolute rates of RNA and protein synthesis. Experiments were carried out in different growth media and with two different strains of E. coli, B/r and K-12. The results indicated: 1) increased rrn gene dosage from either intact or defective rrn genes reduced bacterial growth rates and ribosome activity (protein synthesis rate/average ribosome), and increased rs/rt. 2) Extra intact, but not extra defective, plasmid-borne rrn genes caused the level of ppGpp to be increased in comparison to the pBR322-carrying control strain. 3) As a function of ppGpp, rs/rt was increased with either intact or defective rrn genes. 4) The rRNA synthesis rate/rrn gene was reduced in the presence of extra rrn genes; this reduction in gene activity was greater with intact than with defective rrn genes. An analysis of these results showed that they are consistent with the ppGpp hypothesis of rRNA control but not with a feedback effector role of translating ribosomes.     

The organization of two rRNA (rrn) operons of the slow-growing pathogen Mycobacterium celatum provides key insights into mycobacterial evolution

The slow-growing Mycobacterium celatum is known to have two different 16S rRNA gene sequences. This study confirms the presence of two rrn operons and describes their organization. One operon (rrnA) was found to be located downstream from murA and the other (rrnB) was found downstream from tyrS. The promoter regions were sequenced, and also the intergenic transcribed spacer (ITS1 and ITS2) regions separating the 16S rRNA, 23S rRNA and 5S rRNA gene coding regions. Analysis of the RNA fraction revealed that rrnA is regulated by two (P1 and PCL1) promoters and rrnB is regulated by one (P1). These data show that the two rrn operons of M. celatum are organized in the same way as the two rrn operons of classical fast-growing mycobacteria. This information was incorporated into a phylogenetic analysis of the genus based on both 16S rRNA gene sequences and (where possible) the number of rrn operons per genome. The results suggest that the ancestral Mycobacterium possessed two (rrnA and rrnB) operons per genome and that subsequently, on two separate occasions, an operon (rrnB) was lost, leading to two clusters of species having a single operon (rrnA); one cluster includes the classical pathogens and the other includes Mycobacterium abscessus and Mycobacterium chelonae.     

Toxic effects of high levels of ppGpp in Escherichia coli are relieved by rpoB mutations.

A controversy has surrounded the questions of whether or not guanosine tetraphosphate (ppGpp) is a specific inhibitor of bacterial rRNA and tRNA synthesis, especially during normal exponential growth, and whether the RNA polymerase is the target of ppGpp action. To answer these questions, a pBR322-derived plasmid, pKT28, was constructed that carries the Escherichia coli relA gene encoding a ppGpp synthetase under control of the lacUV5 promoter. The plasmid was used to transform the ppGpp reporter strain VH271 in which expression of beta-galactosidase from an rrnB P1 promoter is inhibited by ppGpp. In the presence of high concentrations of lac inducer, bacteria of the transformed strain accumulate ppGpp with the result that synthesis of rRNA and beta-galactosidase is inhibited and growth ceases. At low concentrations of inducer, growth is only reduced and cells form small white colonies on X-gal indicator plates. After continued incubation, these colonies form blue sectors of faster growing mutant cells. Phage P1 transduction experiments showed that these mutants have mutations cotransducing with rpoB, the gene encoding the beta-subunit of RNA polymerase. One particular mutant strain, KT13, had acquired partial resistance to ppGpp inhibition of rRNA synthesis. The mutation in this strain was cloned by in vivo recombination into an rpoB plasmid. The presence of this plasmid conferred increased resistance to overproduction of ppGpp. These results suggest that ppGpp is a specific inhibitor of rRNA synthesis, even in the absence of amino acid starvation, and that RNA polymerase is involved as the target of ppGpp action.     

Characterization of an rRNA operon (rrnB) of Mycobacterium fortuitum and other mycobacterial species: implications for the classification of mycobacteria.

Mycobacteria are thought to have either one or two rRNA operons per genome. All mycobacteria investigated to date have an operon, designated rrnA, located downstream from the murA gene. We report that Mycobacteriun fortuitum has a second rrn operon, designated rrnB, which is located downstream from the tyrS gene; tyrS is very close to the 3' end of a gene (3-mag) coding for 3-methylpurine-DNA-glycosylase. The second rrn operon of Mycobacterium smegmatis was shown to have a similar organization, namely, 5' 3-mag-tyrS-rrnB 3'. The rrnB operon of M. fortuitum was found to have a single dedicated promoter. During exponential growth in a rich medium, the rrnB and rrnA operons were the major and minor contributors, respectively, to pre-rRNA synthesis. Genomic DNA was isolated from eight other fast-growing mycobacterial species. Samples were investigated by Southern blot analysis using probes for murA, tyrS, and 16S rRNA sequences. The results revealed that both rrnA and rrnB operons were present in each species. The results form the basis for a proposed new scheme for the classification of mycobacteria. The approach, which is phylogenetic in concept, is based on particular properties of the rrn operons of a cell, namely, the number per genome and a feature of 16S rRNA gene sequences.