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The relA gene of Escherichia coli encodes guanosine 3',5'-bispyrophosphate (ppGpp) synthetase I, a ribosome-associated enzyme that is activated during amino acid starvation. The stringent response is thought to be mediated by ppGpp. Mutations in relA are known to result in pleiotropic phenotypes. We now report that three different relA mutant alleles, relA1, relA2, and relA251::kan, conferred temperature-sensitive phenotypes, as demonstrated by reduced plating efficiencies on nutrient agar (Difco) or on Davis minimal agar (Difco) at temperatures above 41[degrees]C. The relA-mediated temperature sensitivity was osmoremedial and could be completely suppressed, for example, by the addition of NaCl to the medium at a concentration of 0.3 M. The temperature sensitivities of the relA mutants were associated with decreased thermotolerance; e.g., relA mutants lost viability at 42[degrees] C, a temperature that is normally nonlethal. The spoT gene encodes a bifunctional enzyme possessing ppGpp synthetase and ppGpp pyrophosphohydrolase activities. The introduction of the spoT207::cat allele into a strain bearing the relA251::kan mutation completely abolished ppGpp synthesis. This ppGpp null mutant was even more temperature sensitive than the strain carrying the relA251::kan mutation alone. The relA-mediated thermosensitivity was suppressed by certain mutant alleles of rpoB (encoding the [beta] subunit of RNA polymerase) and spoT that have been previously reported to suppress other phenotypic characteristics conferred by relA mutations. Collectively, these results suggest that ppGpp may be required in some way for the expression of genes involved in thermotolerance.

Research has been conducted on the Escherichia coli RelA protein activity regulation. Results indicate that the N-terminal of the ribosomal protein L11 is involved in the RelA regulation.

Results show that Escherichia coli cytotoxin RelE interacts with its specific antidote RelB as indicated by their copurification and a positive assay on the yeast two-hybrid assay with the relB and relE genes. Also, purified RelE protein binds to ribosomes.

The temperature sensitivity of the lysis reaction to beta-lactam antibiotic observed in Escherichia coli deprived of amino acids is not due to the heat shock response as reported by previous studies. Mutation studies revealed that E. coli strains that lack the heat shock response gene exhibited similar temperature sensitivity to beta-lactam induced lysis as wild-type E. coli. The temperature sensitivity of the lysis response may be due to direct thermoinactivation of a peptidoglycan hydrolase.

Previous studies have reported that Escherichia coli accumulate guanosine 3',5'-bispyrophosphate (ppGpp) and in consequence, develop penicillin tolerance, as a stringent response under conditions of amino acid deprivation. During the stringent response, synthesis of peptidoglycan and penicillin-induced lysis are inhibited in the amino acid-deprived cells. In a recent study of E. coli, peptidoglycan synthesis and penicillin tolerance were shown to result from the inhibition of phospholipid synthesis and not from the production of ppGpp as earlier suggested. This indicates that peptidoglycan metabolism is dependent on phospholipid synthesis during growth of E. coli.

Guanosine 3',5'-bispyrophosphate (ppGpp) directly correlates with penicillin tolerance in Escherichia coli when overexpressed as demonstrated through ppGpp overexpression in a cloned relA gene without amino acid deprivation. Ongoing phospholipid synthesis is necessary for peptidoglycan metabolism, the inhibition of which causes penicillin tolerance to develop. ppGpp is directly linked to the inhibition of phospholipid synthesis and therefore to penicillin tolerance.

Inhibition of the beta-lactam-induced lysis of amino acid-deprived bacteria upon relaxation of stringent response reveals the dependence of this lysis on phospholipid synthesis. Two stages, the priming stage and the beta-lactam-independent lysis induction stage, involve in the lysis process during amino acid deprivation. Phospholipid synthesis is essential for both the stages. Inhibition of phospholipid synthesis leads to inhibition of peptidoglycan synthesis and the penicillin tolerance associated with the stringent response.

The lytF gene of Escherichia coli, located at 62.5 min on the genetic linkage map, is possibly related to murH, lytD and lytE genes as indicated by phenotypic parallels and common properties of extragenic suppressors. Thermosensitivity and lytF-mediated lysis are suppressed by smhA1 and smhB1 mutations which are extragenic suppressors of thermosensitive mutation. New alleles of smhA and smhB with suppressive properties are isolated.

The lytB gene of Escherichia coli encodes a protein that contributes in the regulation of guanosine 3'5'-bispyrophosphate metabolism. The gene is also involved in temperature sensitivity as well as penicillin tolerance. A fragment of the lytB gene is used as a probe to determine the occurrence of similar genes in other bacteria. Southern blot studies revealed that similar genes are present among gram-negative bacteria.

The identification of the Escherichia coli lytB gene as a previously described openreading frame, designated orf316, in the ileS-lsp operon was reported. The lytBgene is involved in penicillin tolerance and control of the stringent response upon shift to the restrictive temperature. The locus maps at 0.4 min on the linkage map. It was found that the products of the ileS, lsp and lytB genes arenot functionally related, although they are conserved in operon organization.

Examines the temperature-sensitive growth and decreased thermotolerance associated with relA gene mutations in Escherichia coli. Types of relA mutant alleles; Suppression of the relA-mediated thermosensitivity; Osmoremediation of relA-mediated temperature-sensitive growth.

Studies the involvement of the N terminus of ribosomal protein L11 in regulation of the RelA protein of Escherichia coli. Effects of N-terminal deletion fragment of L11 protein on stringent response and accumulation of ppGpp; Influence of proline-rich helix of L11 on RelA regulation; Effect of 'L11 on RelA-ribosome interaction.

Provides information on a study of the temperature-sensitive penicillin reported in amino acid-deprived Escherichia coli. Information on the role of the heat shock response in temperature-dependent tolerance to ...-lactam antibiotics; Detailed information on the effect of temperature on priming and lysis induction stages of ampicillin-induced lysis; Conclusion reached for the basis of temperature sensitivity.

Lipopolsaccharide was isolated from a phage-selected mutant of a wild strain of Aeromonas salmonicida by the aqueous phenol method. The lipopolysaccharide consisted of the R form, containing per mode, three moles of L-glycero-D-manno-heptopyranose, one mole of 3-deoxy-D-manno-2-octulosonic acid (dOclA) and lipid A. The dOclA was not fully assayable by the thiobarbituric acid methods usually used, but its degradation product was detected, after Smith degradation of lipopolysaccharide, either as free 3-deoxy-2-heptulosonic acid (after hydrolysis) or substituted by a mannopyranosyl residue derived from heptose. Mas spectrometry indicated that the dOclA existed in the furanose form and was substituted by the heptose triasaccharide through position six. Methylation analysis, chemical degradation, chromium trioxide oxidation and nuclear magnetic resonance spectroscopy were used to identify the structure of the core oligosaccharide as: LαDHepp(1→2)LαDHepp(1→3)LαD-Hepp(1→6)dOclAf(2→. [ABSTRACT FROM AUTHOR]