Clustered arg genes on a BamHI segment of the Escherichia coli chromosome.

BamHI cleavage of DNAs from transducing phages gamma darg13 (ppc, argECBH, bfe), gamma darg14 (ppc, argECBH) and gamma darg23 (argECBH) yields three purely gamma DNA segments (and, in one case, a fourth), as well as several Excherichia coli-DNA-containing segments. The length (in kilobases, kb) of the segments, determined by electron microscopy and ararose gel electrophoresis is 4.2, 7.5, 8.4, 6.2, 6.9, and 6.4 kb for gamma darg13; 13.0, 7.5, 4.7, 6.2, 6.9, and 6.4 kb for gamma darg 14: and 5.3, 11.0, 4.7, 6.2, 6.9, and 6.4 kb for gamma darg23. Ordering of the segments (in relation to the gamma genetic map and with the direction from left to right corresponding to the clockwise orientation of the E. coli genetic map and to each of the numerical sequences given) reveals, on 26 kb of bacterial DNA, two cleavage sites defining the 7.5-kb segment obtainable from the DNA of either gamam darg13 or gamma darg14. These and analogous findings with argEC and argCB deletion-bearing strains, together with results from heteroduplex experiments, locate argE, argC, argB, and presumably argH on the 7.5-kb segment.     

Regulation of polyamine and streptomycin transport during stringent and relaxed control in Escherichia coli.

Inhibition of polyamine uptake was observed during amino acid depletion in a stringent strain of Escherichia coli CP78 but not in a relaxed strain (CP79). Chloramphenicol was shown partially to relieve the inhibition of uptake. Stringent cells which were induced for a transport system common to both polyamines and streptomycin were found to restrict the uptake of spermidine as well as streptomycin.     

Relaxation effect of chloramphenicol on the stringent control in Escherichia coli.

In 10B601 (rel+) strain possessing a temperature-sensitive valyl-tRNA synthetase, chloramphenicol prevented the formation of guanosine-3'-diphosphate-5'-diphosphate (ppGpp) as well as the stringent control of stable RNA synthesis, under the conditions where the incorporation of valine into protein was still detectable i.e. at the lower restrictive temperatures. On the other hand, the effect of chloramphenicol was not observed at higher restrictive temperatures above 42 degrees C where the incorporation of valine was completely absent. Pretreatment of 10B601 cells with chloramphenicol before transfer to a high restrictive temperature (43.5 degrees C) did retard the onset of accumulation of ppGpp after the shift-up. Duration of the lag period was dependent on the concentration of chloramphenicol added. In parallel with the inability of the cells to accumulate ppGpp, stable RNA synthesis was permitted to continue at that high temperature. These results suggest that chloramphenicol traps aminoacyl-tRNA at the A-sites of ribosomes by damming-up the small flow of aminoacyl-tRNA under the restrictive supply of amino acids. Unchanged tRNA which has been located at the A-site is replaced by the charged one, thus resulting in the suppression of ppGpp formation and in the restoration of stable RNA synthesis.     

Polyamine regulation of stringent control in a polyamine-auxotrophic strain of Escherichia coli.

Escherichia coli BGA8 , a mutant unable to synthesize putrescine, behaves as stringent or relaxed according to the presence or absence of polyamine, respectively, in the culture medium. The relaxed synthesis of RNA can be reverted back to stringent by addition of putrescine or spermidine. The stringent response depends on the concentration of the polyamine in the culture medium. The formation of guanosine 3'-diphosphate 5'-diphosphate elicited by amino acid starvation is stimulated at least 40-fold in putrescine-supplemented bacteria and only about 2-fold in putrescine-depleted cells.     

3H-thymidine incorporation following isoleucine limitation in stringent and relaxed controlled strains of Escherichia coli

Stringent and relaxed strains of E. coli subjected to isoleucine starvation were examined by follow-wing the incorporation of 3H-thymidine into chromosomal DNA. After valine treatment to trigger an isoleucine deprivation (p)ppGpp is synthesized in the stringent strain only. Remarkable differences in the morphology of the amino acid starved cells of the stringent and relaxed strains can be observed. Upon isoleucine limitation 3H-thymidine incorporation into DNA is reduced in both strains, but this inhibition is remarkably delayed in the relaxed strain. Our result show that the reduction of chromosomal DNA synthesis during amino acid limitation occurs also without ppGpp, but in the presence of ppGpp this process is accelerated.     

High-level expression of genes under control of cro translation initiation signals in Escherichia coli

The expression of several genes in Escherichia coli under the control of the lambda p_R promoter and translation initiation signals of the lambda cro gene were studied. Fusions were made in frame at the initiation codon and/or with 5′ translated cro fragments. Expression fluctuated strongly when genes were fused directly at the ATG, whereas constructs, which encode hybrid genes that include at least the first nine codons of the cro gene, always directed high-level synthesis. These fusion proteins were mainly intracellularly precipitated. Our results confirm the poor reliability of ATG vectors for the expression of cloned genes. On the other hand, useful levels of expression are obtained when genes are fused to 5′ cro coding sequences, presumably due to an efficient ribosome binding site configuration.     

Control of arg gene expression in Salmonella typhimurium by the arginine repressor from Escherichia coli K-12

Summary The regulation of synthesis of arg enzymes in Salmonella typhimurium by the arginine repressor of Escherichia coli K-12 has been reevaluated using a strain of S. typhimurium in which the argR gene was rendered nonfunctional by inserting the translocatable tetracyclineresistance element Tn10 into the argR gene. In contrast to previous studies, the introduction of the argR ⁺ allelle of E. coli on an F-prime factor to the argR::Tn10 S. typhimurium strain reduced the synthesis of arg enzymes to essentially wild-type levels. The elevated levels of arg enzymes observed in other hybrid merodiploids may have been the consequence of the formation of hybrid repressor molecules. The readily scoreable phenotype of tetracycline resistance facilitated establishing linkage of cod and argR (0.6% cotransduction) by P22 phage-mediated transduction.     

Control of membrane lipid synthesis in Escherichia coli during growth and during the stringent response.

The regulation of phospholipid synthesis in cells of Escherichia coli was studied in vivo during growth and during the stringent response to amino acid starvation. Strains harboring the hybrid plasmid pLC44-14 (Clark, L., and Carbon, J. (1976) Cell 9, 91-99), which had increased levels of glycerophosphate acyltransferase, were used to study the involvement of this enzyme in the control of phospholipid synthesis. In addition, regulation was studied by measuring the levels of three early intermediates of phospholipid synthesis:phosphatidic acid, CDP-diglyceride, and dCDP-diglyceride. The liponucleotides were measured by a new enzymatic method which allows determinations to be made on crude lipid extracts. Results from experiments on growing cells are consistent with regulation of membrane lipid synthesis occurring in fatty acid synthesis or at the level of glycerophosphate acylation, but not at any later step. Experiments on the inhibition of lipid synthesis during the stringent response make it possible to rule out explanations which involve the inhibition of a single enzyme; enzymes both before and after the liponucleotides in phospholipid synthesis must be affected.     

The arginine repressor of Escherichia coli.

This review tells the story of the arginine repressor of Escherichia coli from the time of its discovery in the 1950s until the present. It describes how the research progressed through physiological, genetic, and biochemical phases and how the nature of the repressor and its interaction with its target sites were unraveled. The studies of the repression of arginine biosynthesis revealed unique features at every level of the investigations. In the early phase of the work they showed that the genes controlled by the arginine repressor were scattered over the linkage map and were not united, as in other cases, in a single operon. This led to the concept of the regulon as a physiological unit of regulation. It was also shown that different alleles of the arginine repressor could result in either inhibition of enzyme formation, as in E. coli K-12, or in stimulation of enzyme formation, as in E. coli B. Later it was shown that the arginine repressor is a hexamer, whereas other repressors of biosynthetic pathways are dimers. As a consequence the arginine repressor binds to two palindromic sites rather than to one. It was found that the arginine repressor not only acts in the repression of enzyme synthesis but also is required for the resolution of plasmid multimers to monomers, a completely unrelated function. Finally, the arginine repressor does not possess characteristic structural features seen in other prokaryotic repressors, such as a helix-turn-helix motif or an antiparallel beta-sheet motif. The unique features have sustained continuous interest in the arginine repressor and have made it a challenging subject of investigation.     

Effects of some antibiotics on the stringent control of RNA synthesis in Escherichia coli.

Effects of neomycin, spectinomycin, tetracycline and chloramphenicol on the stringent control RNA synthesis and on ppGpp synthesis in the rel+-cells of Escherichia coli having a temperature-sensitive valyl-tRNA synthetase were examined. Without antibiotics, ppGpp began to accumulate and both RNA and protein syntheses were inhibited by transferring the exponentially growing cells from 30 degrees C (permissive temp.) to 40 degrees C (non-permissive temp.). Tetracycline or chloramphenicol, when added after the temperature shift, caused a resumption of RNA synthesis and decay of the accumulated ppGpp, while neomycin or spectinomycin had little effect both on RNA synthesis and the level of ppGpp. When the cells were treated with these antibiotics at permissive temperature, the shift of the temperature to 40 degrees C caused neither inhibition of RNA synthesis nor an accumulation of ppGpp. When neomycin or spectinomycin was added at the beginning of the temperature shift, RNA synthesis continued with an accumulation of ppGpp. Tetracycline or chloramphenicol had no such effect under the same conditions; RNA synthesis continued without an accumulation of ppGpp.