I-like R plasmids promote degradation of stable ribonucleic acid in Escherichia coli.

Some I-like R plasmids, R483, R144, R64drd-11 and R621a, belonging to compatibility groups IncI alpha and I gamma promoted degradation of stable RNA in the srnA1 cells of Escherichia coli after addition of rifampin at 42 degrees C. R16 and R834 plasmids of compatibility group IncB also promoted the degradation of RNA. However, other many kinds of plasmids did not. The promotion of RNA degradation was delayed by the addition of chloramphenicol with rifampin.     

Agglutination of bacterial spheroplasts: agglutination-dependent degradation of Escherichia coli ribosomal ribonucleic acid

When Escherichia coli spheroplasts made by ethylenediaminetetraacetic acid and lysozyme were agglutinated by concanavalin A (con A), the degradation of ribosomal ribonucleic acid (rRNA) was found to occur proportionally to the degree of the agglutination, which was determined by microscopic examination or by a newly devised assay based on the slower settling of aggregates. Methyl-alpha-d-glucoside, low temperature or alkaline pH, all of which reverse the agglutination, also reduced the extent of rRNA degradation. This degradation was not due to the direct action of con A since a similar relationship was found in the case of spontaneous agglutination with concentrated spheroplasts in the absence of con A. The possible importance of a change in the cell membrane associated with the agglutination process is discussed in connection with the initiation of rRNA degradation.     

Induction by mercuric ion of extensive degradation of cellular ribonucleic acid in Escherichia coli

Low concentrations of HgCl(2) were found to induce extensive degradation of ribonucleic acid (RNA) in exponentially growing Escherichia coli cells but not in stationary-phase cells. Whereas 80% of cellular RNA was degraded during 90 min of incubation with 10(-5)m HgCl(2) at 37 C, HgCl(2) caused only slight degradation in stationary cells, even when present at concentrations higher than 5 x 10(-5)m. Inhibition of RNA synthesis occurred at almost the same concentration of HgCl(2) as degradation, and the ability of stationary-phase cells to synthesize RNA was also resistant to HgCl(2). The transition of cells from complete sensitivity to HgCl(2) to a fully insensitive state took place simultaneously with the cessation of growth. p-Chloromercuribenzoate was also found to induce remarkable degradation of RNA. In E. coli Q13, a mutant deficient for ribonuclease I, no degradation of RNA was evident, even in the exponential growth phase. 3'-Mononucleotides but not 5'-mononucleotides were found among the degradation products of cellular RNA. 2',3'-Cyclic mononucleotides were produced when RNA was degraded by the cell-free extracts of the Hg treated cells. Almost complete unmasking of the latent ribonuclease occurred in the particle fraction containing subribosomal particles of the Hg-treated cells. These data suggest that the incubation of exponentially growing E. coli cells with HgCl(2) led to the unmasking of ribonuclease I, which resulted in the extensive degradation of cellular RNA. The activation of ribonuclease by HgCl(2) in the isolated particulate fraction of E. coli K-12 which occurred in vitro suggested the presence of an Hg-sensitive inhibitor for ribonuclease I.     

Genetic mapping of the F plasmid gene that promotes degradation of stable ribonucleic acid in Escherichia coli.

F+ Escherichi coli cells that contain an srnA mutant allele degrade their stable ribonucleic acid (RNA) extensively after RNA synthesis is blocked at 42 degrees C. The relevant gene promoting degradation of stable RNA, srnB+, or its promoter was mapped between 1.7 and 2.8 kilobases on the F plasmid by using deleted F' plasmids and chimeric plasmids composed of pSC101 and fragments of F plasmid.     

Role of aminoacyl-transfer ribonucleic acid in the regulation of ribonucleic acid synthesis in Escherichia coli

Morris, D. W. (University of California, San Diego), and J. A. DeMoss. Role of aminoacyl-transfer ribonucleic acid in the regulation of ribonucleic acid synthesis in Escherichia coli. J. Bacteriol. 90:1624-1631. 1965.-A leucine auxotroph of Escherichia coli was examined for its rate of ribonucleic acid (RNA) synthesis and the level of charged leucine-, arginine-, and valine-specific transfer RNA (tRNA) during the exponential growth period and when growth was limited by leucine starvation. During the logarithmic growth period, the leucine-specific tRNA was 70% charged, arginine-specific tRNA was 30% charged, and the valine-specific tRNA was 80% charged. When leucine became limiting, RNA synthesis was inhibited and the levels of charged arginine- and valine-specific tRNA remained constant, whereas the level of charged leucine-specific tRNA dropped to 40%. Examination of the leucyl-tRNA during the leucine starvation period showed that this 40% level is maintained by protein turnover. Addition of chloramphenicol or puromycin to a leucine-starved culture derepressed RNA synthesis. In the presence of chloramphenicol, the leucine-specific tRNA was fully charged; however, in the presence of puromycin the amount of charged leucine-specific tRNA remained at the starved level. Therefore, during leucine starvation the level of uncharged leucine-specific tRNA is not invariably correlated with the rate of RNA synthesis. We propose that it is the availability of charged tRNA and not the amount of uncharged tRNA which is the important factor in the amino acid control of RNA synthesis.     

Organization of transfer ribonucleic acid genes in the Escherichia coli chromosome.

The arrangement of transfer ribonucleic acid (RNA) genes in the chromosome of Escherichia coli K-12 (C600) was examined with the techniques of restriction endonuclease digestion and Southern blotting. The number and size of restriction fragments containing transfer or ribosomal RNA sequences or both were estimated by a variety of restriction endonucleases, including EcoRI, BglI, SmaI, SalI, BamHI, and PstI. EcoRI liberated a minimum of 27 fragments which hybridized to transfer RNA and 16 which hybridized to ribosomal RNA. Enzymes which did not cut within the ribosomal RNA operons (PstI and BamHI) liberated 16 and 13 fragments, respectively, which hybridized to transfer RNA. Five PstI and six BamHi fragments also hybridized to ribosomal RNA, suggesting that there may be at least 11 chromosomal locations distinct from ribosomal RNA operons which encode transfer RNA genes. In addition, our data indicated that several transfer RNA genes may be very close to the 5' proximal ends of certain ribosomal RNA operons and close to the 3' distal ends of all seven ribosomal RNA operons. Similar studies have been carried out with 22 purified species of transfer RNA, and we report here the number and size of EcoRI restriction fragments which hybridize to these transfer RNA species.     

Galactose-specific messenger ribonucleic acid contents in Escherichia coli

A method is described for measuring the proportion of galactose-specific mRNA (gal-mRNA) in the total RNA extracted from pulse-labelled cells of Escherichia coli K12, by DNA-RNA hybridization with DNA prepared from bacteriophage lambdadg. RNA from wild-type E. coli was compared with RNA from a homogenote carrying the gal operon both in the chromosome and in a substituted sex-factor, and with RNA from a deletion strain that carried the galactose operon only in the exogenote. In each case the cultures were induced with fucose. Under these conditions the amount of gal-mRNA was found to be proportional to the content of galactokinase in the different cultures, and to the gene frequency. The amounts of gal-mRNA in an O(c) mutant and an R(-) mutant were also proportional to the observed contents of galactokinase. In cultures repressed for the enzymes of the galactose operon with thiomethylgalactoside, the content of gal-mRNA was higher than expected from the content of galactokinase. Possible explanations of this finding are discussed.     

Multiple forms of lysyl-transfer ribonucleic acid synthetase in Escherichia coli.

Lysyl-transfer ribonucleic acid synthetase (EC 6.1.1.6) was identified as four polypeptide spots after two-dimensional polyacrylamide gel electrophoresis of whole-cell lysates of Escherichia coli. Identification was made by migration with partially purified enzyme preparations, by peptide map patterns, by mutant analysis, and by correlation of spot intensities with changes in enzyme levels under different growth conditions. Wild-type cells growing at 37 degrees C in glucose minimal medium displayed the enzyme predominantly as two spots (spots I and III). Growth at 46 degrees C, growth in the presence of alanine or glycyl-L-leucine, or growth of a strain with a mutational deficiency in S-adenosylmethionine synthetase (metK) greatly increased the synthesis of two other spots (spots II and IV). Polypeptides I and III, but not polypeptides II and IV, had altered isoelectric points in a lysyl-transfer ribonucleic acid synthetase mutant. These data suggest that multiple forms of lysyl-transfer ribonucleic acid synthetase exist in vivo and that they may be encoded by more than one gene.