Stringent control of peptidoglycan biosynthesis in Escherichia coli K-12.

[3H]Diaminopimelic acid (Dap) was incorporated exclusively into peptidoglycan by Escherichia coli strains auxotrophic for both lysine and Dap. The rate of [3H]Dap incorporation by stringent (rel+) strains was significantly decreased when cells were deprived of required amino acids. The addition of chloramphenicol to amino acid-starved rel+ cultured stimulated both peptidoglycan and ribonucleic acid synthesis. In contrast, a relaxed (relA) derivative incorporated [3H]Dap at comparable rates in the presence or absence of required amino acids. Physiologically significant concentrations of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) inhibited the in vitro synthesis of both carrier lipid-linked intermediate and peptidoglycan catalyzed by a particulate enzyme system. The degree of inhibition was dependent on the concentration of ppGpp in the reaction mixture. Thus, the results of in vivo and in vitro studies indicate that peptidoglycan synthesis is stringently controlled in E. coli.     

Involvement of the relA gene in the autolysis of Escherichia coli induced by inhibitors of peptidoglycan biosynthesis

It is generally assumed that inhibitors of peptidoglycan biosynthesis do not kill nongrowing bacteria. An exceptional case is reported here. The addition of chloramphenicol to amino acid-deprived cultures of relA+ strains of Escherichia coli which were treated with beta-lactam antibiotics, D-cycloserine, or moenomycin resulted in lysis. This phenomenon is termed chloramphenicol-dependent lysis. To be effective, chloramphenicol had to be present at its minimum growth-inhibitory concentration (or higher). Analogs of chloramphenicol which did not bind to ribosomes were completely ineffective. Amino acid deprivation was actually not required to demonstrate chloramphenicol-dependent lysis, and cultures treated with growth-inhibitory levels of chloramphenicol alone were lysed when challenged with inhibitors of peptidoglycan synthesis. Peptidoglycan synthesis has been shown previously to be under stringent (relA+) control, and chloramphenicol is known to be an antagonist of stringent control. Thus, it is proposed that the mechanism of chloramphenicol-dependent lysis is based on the ability of chloramphenicol to relax peptidoglycan synthesis in nongrowing relA+ bacteria. This is also consistent with the observation that treatment of amino acid-deprived relA mutants with inhibitors of peptidoglycan synthesis resulted in lysis, i.e., without the mediation of chloramphenicol.     

Control of lipopolysaccharide biosynthesis and release by Escherichia coli and Salmonella typhimurium.

The influence of the relA gene on lipopolysaccharide (LPS) biosynthesis and release by Escherichia coli and Salmonella typhimurium was investigated. Similar results were obtained with both species. The incorporation of [3H]galactose into LPS by galE mutants was inhibited by at least 50% (as compared with normal growing controls) during amino acid deprivation of relA+ strains. This inhibition could be prevented by the treatment of the amino acid-deprived relA+ bacteria with chloramphenicol, a known antagonist of the stringent control mechanism. Furthermore, LPS biosynthesis was not inhibited during amino acid deprivation of isogenic relA mutant strains. These results indicate that LPS synthesis is regulated by the stringent control mechanism. Normal growing cells of both relA+ and relA strains released LPS into the culture fluid at low rates. Amino acid deprivation stimulated the rate of LPS release by relA mutants but not by relA+ bacteria. Chloramphenicol treatment markedly stimulated the release of cell-bound LPS by amino acid-deprived relA+ cells. Thus, a low rate of LPS release was characteristic of normal growth and could be increased in nongrowing cells by relaxing the control of LPS synthesis.     

Involvement of the relA gene product and feedback inhibition in the regulation of DUP-N-acetylmuramyl-peptide synthesis in Escherichia coli.

The regulation of uridine diphosphate-N-acetylmuramyl-peptide (UDP-MurNAc-peptide) synthesis was studied by labeling Escherichia coli strains auxotrophic for lysine and diaminopimelate with [3H]diaminopimelate for 15 min under various conditions. The amounts of [3H]diaminopimelate incorporated into UDP-MurNAc-tripeptide and -pentapeptide by a stringent (rel+) strain were the same in the presence or absence of lysine. Chloramphenicol-treated rel+ cells showed a 2.8-fold increase in labeled UDP-MurNAc-pentapeptide. An isogenic relaxed (relA) strain deprived of lysine showed a 2.7-fold increase in UDP-MurNAc-pentapeptide. Thus, UDP-MurNAc-pentapeptide synthesis is regulated by the relA gene. D-Cycloserine treatment of rel+ and relA strains caused a depletion of intracellular UDP-MurNAc-pentapeptide. Labeled UDP-MurNAc-tripeptide accumulated in D-cycloserine-treated cells of the rel+ and relA strains, suggesting that UDP-MurNAc-pentapeptide is a feedback inhibitor of UDP-MurNAc-peptide synthesis. In lysine-deprived cells, D-cycloserine treatment caused 41- and 71-fold accumulations of UDP-MurNAc-tripeptide in rel+ and relA strains, respectively. A 124-fold increase in UDP-MurNAc-tripeptide occurred in lysine-deprived rel+ cells treated with both chloramphenicol and D-cycloserine. These results indicate that both the relA gene product and feedback inhibition are involved in regulating UDP-MurNAc-peptide synthesis during amino acid deprivation.     

Beta-lactam-induced bacteriolysis of amino acid-deprived Escherichia coli is dependent on phospholipid synthesis.

The penicillin tolerance of amino acid-deprived relA+ Escherichia coli is attributed to the stringent response; i.e., relaxation of the stringent response suppresses penicillin tolerance. The beta-lactam-induced lysis of amino acid-deprived bacteria resulting from relaxation of the stringent response was inhibited by cerulenin, or by glycerol deprivation in the case of a gpsA mutant (defective in the biosynthetic sn-glycerol 3-phosphate dehydrogenase). Therefore, beta-lactam-induced lysis of amino acid-deprived cells was dependent on phospholipid synthesis. The lysis process during amino acid deprivation can be experimentally dissociated into two stages designated the priming stage (during which the interaction between the beta-lactam and the penicillin-binding proteins occurs) and the beta-lactam-independent lysis induction stage. Both stages were shown to require phospholipid synthesis. It has been known for some time that the inhibition of phospholipid synthesis is among the plethora of physiological changes resulting from the stringent response. These results indicate that the inhibition of peptidoglycan synthesis and the penicillin tolerance associated with the stringent response are both secondary consequences of the inhibition of phospholipid synthesis.     

Inhibition of uridine 5'-diphosphate-N-acetylmuramyl-L-alanine synthetase by beta-chloro-L-alanine in Escherichia coli

The synthesis of the nucleotide precursors for peptidoglycan is regulated by the relA gene in Escherichia coli. Thus, nucleotide precursors labeled with [3H]diaminopimelic acid accumulated in a relA strain but not in an isogenic relA+ strain during amino acid deprivation. Furthermore, nucleotide precursor synthesis was relaxed in the amino acid deprived relA+ strain by treatment with chloramphenicol. Uridine diphosphate-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) was the major component accumulated during the relaxed synthesis of nucleotide precursors in both relA+ and relA strains. The effect of beta-chloro-L-alanine (CLA) on the relaxed synthesis of nucleotide precursors for peptidoglycan was determined. At a low concentration (0.0625 mM) CLA inhibited the synthesis of UDP-MurNAc-pentapeptide and caused the accumulation of UDP-MurNAc-tripeptide. Thus, low concentrations of CLA probably inhibited alanine racemase, as reported previously. Higher concentrations of CLA also inhibited an earlier step in nucleotide precursor synthesis. This was shown to be due to the inhibition of UDP-MurNAc-L-alanine synthetase by CLA. CLA inhibited the activity of this enzyme in cell-free extracts as well as in intact cells.     

Direct correlation between overproduction of guanosine 3',5'-bispyrophosphate (ppGpp) and penicillin tolerance in Escherichia coli.

The penicillin tolerance exhibited by amino acid-deprived Escherichia coli has been previously proposed to be a consequence of the stringent response. Evidence indicating that penicillin tolerance is directly attributable to guanosine 3',5'-bispyrophosphate (ppGpp) overproduction and not to some other effect of amino acid deprivation is now presented. Accumulation of ppGpp in the absence of amino acid deprivation was achieved by the controlled overexpression of the cloned relA gene, which encodes ppGpp synthetase I. The overproduction of ppGpp resulted in the inhibition of both peptidoglycan and phospholipid synthesis and in penicillin tolerance. The minimum concentration of ppGpp required to establish these phenomena was determined to be 870 pmol per mg (dry weight) of cells. This represented about 70% of the maximum level of ppGpp accumulated during the stringent response. Penicillin tolerance and the inhibition of peptidoglycan synthesis were both suppressed when ppGpp accumulation was prevented by treatment with chloramphenicol, an inhibitor of ppGpp synthetase I activation. Glycerol-3-phosphate acyltransferase, the product of plsB, was recently identified as the main site of ppGpp inhibition in phospholipid synthesis (R. J. Health, S. Jackowski, and C. O. Rock, J. Biol. Chem. 269:26584-26590, 1994). The overexpression of the cloned plsB gene reversed the penicillin tolerance conferred by ppGpp accumulation. This result supports previous observations indicating that the membrane-associated events in peptidoglycan metabolism were dependent on ongoing phospholipid synthesis. Interestingly, treatment with beta-lactam antibiotics by itself induced ppGpp accumulation, but the maximum levels attained were insufficient to confer penicillin tolerance.     

relA gene control of the synthesis of lipid A fatty acyl moieties.

The incorporation of [14C]acetate into the fatty acid moieties of lipid A was measured during amino acid starvation of rel+ and relA strains of Escherichia coli K-12. The synthesis of the beta-hydroxymyristate and other fatty acid moieties was inhibited two- to fourfold in rel+ strains, whereas no inhibition was observed in relA strains. The fatty acid compositions of the phospholipids synthesized after amino acid starvation or rel+ and relA strains were also determined.     

Dissociation of the ampicillin-induced lysis of amino acid-deprived Escherichia coli into two stages.

The ampicillin-induced lysis of amino acid-deprived relA+ Escherichia coli was dissociated into two separate stages. The early stage ("priming") requiring the presence of ampicillin apparently involved the interaction of ampicillin with a target penicillin-binding protein. The later stage ("lysis induction") was ampicillin independent and required only chloramphenicol to relax the RelA-dependent control of peptidoglycan hydrolase activity.     

Regulation of peptidoglycan biosynthesis in relA+ and relA- strains of Escherichia coli during diauxic growth on glucose and lactose.

In both relA+ and relA- derivatives, the biosynthesis of peptidoglycan, lipid intermediates, and nucleotide precursors abruptly halted at the onset of diauxic lag from glucose to lactose with a concomitant accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp). These results are consistent with the proposal that ppGpp is involved in inhibiting the incorporation of disaccharide-pentapeptide into peptidoglycan and in regulating nucleotide precursor synthesis.     

Regulation of intracellular protein breakdown in stringent and relaxed strains of E. coli,

Regulation of protein breakdown by amino acids is abolished in relaxed strains of E. coli, but these mutants do respond to the deprivation of inorganic phosphate. Protein synthesis is directly required for the control of protein degradation. We suggest that the failure of amino acid-deprived rel- strains to regulate protein breakdown may be due to defective protein synthesis.     

The relA locus specifies a positive effector in branched-chain amino acid transport regulation.

The regulation of branched-chain amino acid transport and periplasmic binding proteins was studied in Escherichia coli strains which were isogenic except for the relA locus, the gene for the "stringent factor," which is responsible for guanosine tetraphosphate synthesis. The strain containing the relA mutation could not be derepressed for the synthesis of leucine transport or binding proteins when shifted from a medium containing all 20 amino acids in excess to one in which leucine was limiting. The relA+ strain showed normal derepression under these conditions.     

Regulation of D-alanine carboxypeptidase and peptidoglycan cross-linkage in amino acid-deprived Escherichia coli.

The effect of amino acid deprivation on the activities of D-alanine carboxypeptidase (CPase) and peptidoglycan transpeptidase in Escherichia coli was determined. Enzymes were assayed in ether-treated bacteria (ETB) which were permeable to peptidoglycan nucleotide precursors. ETB were prepared at intervals from cultures grown in the presence and absence of a required amino acid. The specific activity of CPase in ETB decreased 50 to 85% during amino acid deprivation. This was paralleled by a 60 to 70% decrease in the specific activity of peptidoglycan transpeptidase. Both enzymes reached their lowest level of activity about 40 min after the onset of amino acid deprivation. The decrease in CPase activity apparently was not due to degradation of the enzyme, since full activity was restored after disruption of ETB by sonication. A decrease in CPase activity was associated with an enhancement of transpeptidation. The peptidoglycan synthesized in vitro by amino acid-deprived ETB was 1.7 times more cross-linked than the peptidoglycan synthesized by control ETB These results support the proposal that CPase may be involved in regulating transpeptidation in E. coli.     

Rates of peptidoglycan turnover and cell growth of Bacillus subtilis are correlated

Peptidoglycan turnover was measured by the decrease of trichloroacetic acid-precipitable label in cells labeled with N-acetyl-D-[14C]glucosamine. The rate of turnover was reduced strongly by the inhibition of RNA or protein synthesis and weakly by the inhibition of lipid, peptidoglycan, or DNA synthesis. It increased with the growth rate (which was controlled by the concentration of oxomethylvalerate limiting the intracellular isoleucine supply) to the same degree in stringent (rel+) and isogenic relaxed (relA) strains. In these and all other strains tested, the turnover rate (k) increased with the growth rate (g) according to the equation, k = 0.70 X g1.38, even when the growth rate was systematically altered by changes in the temperature or in the composition of the medium.     

Temperature sensitivity of the penicillin-induced autolysis mechanism in nongrowing cultures of Escherichia coli.

The effect of incubation temperature on the ampicillin-induced autolysis of nongrowing Escherichia coli was determined. The autolysis mechanisms in amino acid-deprived relA mutant cells treated with chloramphenicol were temperature sensitive. This temperature-sensitive autolysis was demonstrated in three independent ways: turbidimetric determinations, viable cell counts, and solubilization of radiolabeled peptidoglycan.     

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

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 beta-galactosidase upon induction. Analysis of the RNA accumulated in relaxed strains indicated it was present as transfer RNA as well as 50S and 30S 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.     

Influence of amino acid starvation on guanosine 5''-diphosphate 3''-diphosphate basal-level synthesis in Escherichia coli.

We observed that the synthesis of basal-level guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in both relA mutants and relA+ relC strains of Escherichia coli decreased in response to amino acid limitation and that this was accompanied by an increase in ribonucleic acid (RNA) synthesis. Addition of the required amino acid to starved cultures of relaxed bacteria resulted in the resumption of ppGpp synthesis and a concomitant decrease in RNA production. Our results indicate that relA mutants retain a stringent factor-independent ribosomal mechanism for basal-level ppGpp synthesis. They also suggest that in relA+ bacteria, stringent factor-mediated ppGpp synthesis and the production of basal-level ppGpp are mutually exclusive. These findings substantiate the hypothesis that there are two functionally discrete mechanisms for ppGpp synthesis in E. coli. Through these studies we have also obtained new evidence which indicates that ppGpp serves as a modulator of RNA synthesis during balanced growth as well as under conditions of nutritional downshift and starvation.