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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 ␤-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, ␤-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 ␤-lactam and the penicillinbinding proteins occurs) and the ␤-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.

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It is generally assumed that only actively growing bacteria are killed by inhibitors of peptidoglycan synthesis. Several exceptional examples are described here. As expected, ampicillin did not lyse nongrowing, amino acid-deprived cultures of relA+ strains of Escherichia coli, but the subsequent addition of several ribosome inhibitors (chloramphenicol, tetracycline, gentamicin, and kanamycin) caused various degrees of lysis in such ampicillin-treated cultures. Of the antibiotics tested, only streptomycin was ineffective in this regard. Peptidoglycan synthesis has been shown to be inhibited in amino acid-deprived reLAU bacteria by the stringent control mechanism (E. E. Ishiguro and W. D. Ramey, J. Bacteriol. 127:1119Bacteriol. 127: -1126Bacteriol. 127: , 1976, and the ribosome inhibitors tested here relaxed peptidoglycan synthesis to various degrees under these conditions. The relative lysis-inducing activities of the ribosome inhibitors on ampicillin-treated, amino acid-deprived bacteria were directly correlated to their relative activities as stringent control antagonists. This phenomenon was not dependent on amino acid deprivation. Cultures treated with growth inhibitory levels of the various ribosome inhibitors alone were lysed by ampicillin, apparently because peptidoglycan synthesis continues uninhibited when growth is arrested by treatment with ribosome inhibitors. These results indicate that autolysis can be triggered by the inhibition of peptidoglycan synthesis occurring in the absence of wall expansion; i.e., active cell growth is unnecessary.It is generally assumed that ,3-lactam antibiotics and other inhibitors of peptidoglycan synthesis kill only actively growing bacteria. For example, Escherichia coli cells which are deprived of a required amino acid, or other growth factors, develop tolerance to ,-lactam antibiotics, and this forms the basis for the classic penicillin enrichment technique for the isolation of auxotrophic mutants (3, 8). We recently described an interesting exception which we call chloramphenicol-dependent lysis (7a). In this case, the addition of chloramphenicol to amino acid-deprived cultures of E. coli which were treated with ,-lactam antibiotics, D-cycloserine, or moenomycin resulted in lysis. Furthermore, amino acid deprivation was not essential for this phenomenon; i.e., cultures inhibited with chloramphenicol alone were subsequently lysed when treated with inhibitors of peptidoglycan synthesis.Peptidoglycan synthesis in E. coli is regulated by the stringent control mechanism (6, 7). Thus, amino acid deprivation results in the rapid accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a putative mediator of stringent control (1, 4), and in the concomitant inhibition of peptidoglycan synthesis in relA+ strains. On the other hand, relA mutants do not accumulate ppGpp during amino acid deprivation, and peptidoglycan synthesis continues (relaxed control). We considered three observations to be important in formulating a possible mechanistic basis for chloramphenicol-de...

Escherichia coli murein transglycosylase, a potential autolysin which splits the sugar chains of the murein sacculus, was rapidly purified from a crude cell extract by sequential chromatography on columns of blue Sepharose and poly(U)‐Sepharose. In accordance with the binding to blue Sepharose and poly(U)‐Sepharose, the transglycosylase is inhibited by Cibacron blue F3G‐A, the affinity ligand of blue Sepharose, and also by polynucleotides, the latter, however, with varying efficiency. Among the polynucleotides tested, single‐stranded DNA was found to be one of the most potent inhibitors. When bound to a blue Sepharose column, transglycosylase could be displaced from the column with single‐stranded DNA. Taken together, these results point to a polynucleotide binding area on the transglycosylase molecule. Some aspects of the blue Sepharose affinity chromatography and the possible biological significance of the transglycosylase are discussed.