Effect of the “Ribonucleic Acid Control” Locus in Escherichia coli on T4 Bacteriophage-Specific Ribonucleic Acid Synthesis

Amino acid control of ribonucleic acid (RNA) synthesis in bacteria is known to be governed genetically by the rel locus. We investigated whether the rel gene of the host would also exert its effect on the regulation of phage-specific RNA synthesis in T4 phage-infected Escherichia coli cells. Since T-even phage infection completely shuts off host macromolecular synthesis, phage RNA synthesis could be followed specifically by the cumulative incorporation of radioactivity from labeled precursors into RNA of infected cells. Labeled uracil was shown to accumulate in phage-specific RNA for 30 to 35 min after infection, a phenomenon which probably reflects an expansion of the labile phage-RNA pool. Amino acid starvation was effected by the use of auxotrophic bacterial strains or thienylalanine. The latter substance is an amino acid analogue which induces a chemical auxotrophy by inhibiting the biosynthesis of phenylalanine, tyrosine, and tryptophan. Phage RNA synthesis was strictly dependent on the presence of amino acids, whereas phage deoxyribonucleic acid synthesis was not. By the use of several pairs of bacterial strains which were isogenic except for the rel gene, it was demonstrated that amino acid dependence was related to the allelic state of this gene. If the rel gene was mutated, amino acid starvation did not restrict phage RNA synthesis.     

Escherichia coli strain with a deletion of the chromosomalampC gene marked with TcR, suitable for production of penicillin G acylase

Escherichia coli strain which contains a marker of tetracycline resistance gene (TcR) placed by P1 transduction beside the chromosomal deletion of ampC gene (delta ampC) coding for beta-lactamase was constructed. Such introduction of TcR marker permits a fast and simple selection for the transfer of delta ampC by P1 transduction into industrial E. coli strains. This approach was used for constructing an E. coli strain suitable for penicillin acylase production.     

Stringent control during carbon starvation of marine Vibrio sp. strain S14: molecular cloning, nucleotide sequence, and deletion of the relA gene.

In order to evaluate the role of the stringent response in starvation adaptations of the marine Vibrio sp. strain S14, we have cloned the relA gene and generated relaxed mutants of this organism. The Vibrio relA gene was selected from a chromosomal DNA library by complementation of an Escherichia coli delta relA strain. The nucleotide sequence contains a 743-codon open reading frame that encodes a polypeptide that is identical in length and highly homologous to the E. coli RelA protein. The amino acid sequences are 64% identical, and they share some completely conserved regions. A delta relA::kan allele was generated by replacing 53% of the open reading frame with a kanamycin resistance gene. The Vibrio relA mutants displayed a relaxed control of RNA synthesis and failed to accumulate ppGpp during amino acid limitation. During carbon and energy starvation, a relA-dependent burst of ppGpp synthesis concomitant with carbon source depletion and growth arrest was observed. Also, in the absence of the relA gene, there was an accumulation of ppGpp during carbon starvation, but this was slower and smaller than that which occurred in the stringent strains, and it was preceded by a marked decrease in the [ATP]/[ADP] ratio. In both the wild-type and the relaxed strains, carbon source depletion caused an immediate decrease in the size of the GTP pool and a block of net RNA accumulation. The relA mutation did not affect long-term survival or the development of resistance against heat, ethanol, and oxidative stress during carbon starvation of Vibrio sp. strain S14.     

Effect of amino acid starvation on glucose transport in two archaeal organisms

When protein synthesis is arrested by amino acid starvation, Escherichia coli wild-type strains show stringent control (SC) over stable RNA (sRNA) accumulation as well as a large number of other growth-related processes. One of the events under SC is transport of metabolites. Thus, under amino acid starvation, E. coli fails to accumulate the non-metabolizable glucose analog alpha-methyl-D-glucoside, whereas isogenic relaxed strains continue to take up this glucose analog. Unlike the Bacteria, most wild-type archaeal strains show relaxed control of sRNA accumulation, although a number of stringent strains have been identified. In order to determine whether stringency in the Archaea affects physiological events different from sRNA accumulation, transport of glucose analogs was examined under amino acid starvation in two stringent archaeal strains, Haloferax volcanii and Sulfolobus acidocaldarius. The experiments were performed with 2-deoxy-D-glucose, which was shown to be transported, but metabolized very limitedly. Unlike E. coli, H. volcanii and S. acidocaldarius continued to transport 2-deoxy-D-glucose under amino acid starvation. Thus, in both Archaea glucose analog transport is not under SC, as it is in E. coli.     

Endogenous Dark Respiration of the Blue-Green Alga, Plectonema boryanum

The envA mutation in Escherichia coli K-12, which maps at 1.5 min, was previously shown to mediate sensitivity to gentian violet as well as to several antibiotics. Moreover, strains containing the envA gene were recently found to be lysed by lysozyme in the absence of ethylenediaminetetraacetate. It is here reported that the envA mutation mediates an increased uptake of gentian violet. The uptake of the dye was markedly affected by growth with different antibiotics interfering with macromolecular synthesis. Amino acid starvation of a strain containing envA with a stringent control of ribonucleic acid (RNA) synthesis resulted in a decreased uptake of gentian violet. However, no decrease in dye uptake was found during starvation in an envA transductant with a relaxed control of RNA synthesis. Inhibition of deoxyribonucleic acid (DNA) synthesis by nalidixic acid decreased the uptake of gentian violet of envA cells and, in addition, rendered the cells insensitive to the lytic action of lysozyme. Chloramphenicol treatment increased penetrability in wild-type and starved envA cells. In most instances, this effect of chloramphenicol was prevented by selectively interfering with DNA or RNA synthesis. A coordinate regulation of nucleic acid synthesis and penetrability is suggested.     

Selective retention of recombinant plasmids coding for human insulin

Plasmids may be lost from Escherichia coli K-12 hosts that are cultured without selection for plasmid retention. This is particularly true for chimeric plasmids that incorporate genes for human insulin into vectors derived from pBR322. The cIts857 gene of bacteriophage lambda was inserted into the bla gene of the human-insulin-coding plasmids, pIA7 delta 4 delta 1, pIB7 delta 4 delta 1 and pHI7 delta 4 delta 1, generating the new plasmids pPR17, pPR18 and pPR19, respectively, which produced the thermosensitive lambda repressor. The cI gene was downstream from the pM and pbla promoters, so that it may have been expressed from either or both promoters. Separate E. coli K-12 RV308 host strains containing the new recombinants were lysogenized with the repressor-defective bacteriophage lambda cI90. Loss of the plasmid from the lysogens causes concomitant loss of the lambda repressor and cell death, because the prophage is induced to enter the lytic growth cycle. The system effectively forces retention of the plasmid in all viable cells in the culture.     

Control of rRNA and tRNA syntheses in Escherichia coli by guanosine tetraphosphate

The expression of stable RNA (rRNA and tRNA) genes and the concentration of guanosine tetraphosphate (ppGpp) were measured in an isogenic pair of relA+ and relA derivatives of Escherichia coli B/r. The cells were either growing exponentially at different rates or subject to amino acid starvation when they were measured. The specific stable RNA gene activity (rs/rt, the rate of rRNA and tRNA synthesis relative to the total instantaneous rate of RNA synthesis) was found to decrease from 1.0 at a ppGpp concentration of 0 (extrapolated value) to 0.24 at saturating concentrations of ppGpp (above 100 pmoles per optical density at 460 nm unit of cell mass). The same relationship between the rs/rt ratio and ppGpp concentration was obtained independent of the physiological state of the bacteria (i.e., independent of the growth rate or of amino acid starvation) and independent of the relA allele. It can be concluded that ppGpp is an effector for stable RNA gene control and that stable RNA genes are not controlled by factors other than the ppGpp-mediated system. The results were shown to be qualitatively and quantitatively consistent with data on in vitro rRNA gene control by ppGpp, and they were interpreted in the light of reported ideas derived from those in vitro experiments.     

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.