Ribonucleic Acid Regulation in Amino Acid-Limited Cultures of Escherichia coli Grown in a Chemostat

Abstract

The regulation of ribonucleic acid (RNA) synthesis was examined in cultures of bacteria whose growth was limited in the chemostat by the supply of a required amino acid. Strains possessing the relaxed ( relA ) mutation accumulated excess RNA (relative to protein) at low growth rates when growth was limited by arginine, histidine, or cysteine but not when limited by methionine. In contrast, stringent ( relA + ) strains maintained a constant RNA/protein ratio with decreasing growth rate regardless of the amino acid used to limit growth. The presence of excess RNA in relaxed strains was accompanied by an absence of increase in RNA production upon addition of chloramphenicol, a lag upon shift-up in growth by addition of excess of the limiting amino acid, and a decreased rate of production of β-galactosidase upon induction. Analysis of the RNA accumulated in relaxed strains indicated it was present as transfer RNA as well as 50 S and 30 S ribosomal subunits. Microscope examination of the relaxed strains during histidine-, arginine-, or cysteine-limited growth in the chemostat showed them to be 10 to 20 times longer in size than the stringent strains. Also, cell density was reduced to one-tenth when the increased size was observed. An analysis of the amount of ppGpp present in all slow-growing amino acid-limited cultures (relaxed and stringent) demonstrated that only basal levels of ppGpp were made. These data are consistent with the hypothesis that when growth is limited in the chemostat by an initiation event in protein synthesis, i.e., limited methionine, RNA regulation occurs in relaxed as well as stringent strains. Also, when other amino acids are limiting in concentration during translation, errors occur in relaxed strains, resulting in misread proteins.