Involvement of the N terminus of ribosomal protein L11 in regulation of the RelA protein of Escherichia coli.

Amino acid-deprived rplK (previously known as relC) mutants of Escherichia coli cannot activate (p)ppGpp synthetase I (RelA) and consequently exhibit relaxed phenotypes. The rplK gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA. To investigate the role of L11 in the stringent response, a derivative of rplK encoding L11 lacking the N-terminal 36 amino acids (designated 'L11) was constructed. Bacteria overexpressing 'L11 exhibited a relaxed phenotype, and this was associated with an inhibition of RelA-dependent (p)ppGpp synthesis during amino acid deprivation. In contrast, bacteria overexpressing normal L11 exhibited a typical stringent response. The overexpressed 'L11 was incorporated into ribosomes and had no effect on the ribosome-binding activity of RelA. By several methods (yeast two-hybrid, affinity blotting, and copurification), no direct interaction was observed between the C-terminal ribosome-binding domain of RelA and L11. To determine whether the proline-rich helix of L11 was involved in RelA regulation, the Pro-22 residue was replaced with Leu by site-directed mutagenesis. The overexpression of the Leu-22 mutant derivative of L11 resulted in a relaxed phenotype. These results indicate that the proline-rich helix in the N terminus of L11 is involved in regulating the activity of RelA.

Purification of the RelB and RelE proteins of Escherichia coli: RelE binds to RelB and to ribosomes.

The direct interaction of the Escherichia coli cytotoxin RelE with its specific antidote, RelB, was demonstrated in two ways: (i) copurification of the two proteins and (ii) a positive yeast two-hybrid assay involving the relB and relE genes. In addition, the purified RelE protein exhibited ribosome-binding activity in an in vitro assay, supporting previous observations suggesting that it is an inhibitor of translation.

Dimerization of the RelA protein of Escherichia coli.

The RelA protein of Escherichia coli is a ribosome-associated (p)ppGpp synthetase that is activated by amino acid deprivation. It was recently reported that the activity of RelA is regulated by oligomerization mediated by the C-terminal domain of RelA. The oligomerization of RelA is further characterized in this study. The C-terminal domain consisting of amino acids 455-744, designated 'RelA, formed homooligomers as well as heterooligomers with RelA as demonstrated by copurification of RelA and 'RelA and by an affinity blotting assay. Glutaraldehyde-induced cross-linking indicated that the oligomer was a dimer. The functional analysis of 'RelA was based on a combination of yeast two-hybrid analysis, the determination of the effects of overexpression of 'RelA derivatives on the stringent response, and the cellular localization of the overexpressed 'RelA derivatives. These studies indicated that two regions, designated 'RelA-1 (amino acids 455-538) and 'RelA-2 (amino acids 550-682), were involved in dimerization. The involvement of one of these two regions, RelA-2, is consistent with a previous site-directed mutagenesis study. In addition to dimerization, 'RelA-2 apparently contained the main ribosome-binding domain of RelA. The third region, 'RelA-3 (amino acids 682-744), was not involved in either dimerization or ribosome binding. The overexpression of 'RelA-1 and 'RelA-2, but not 'RelA-3, inhibited the stringent response. These results support the previously proposed model which suggests a role for oligomerization in the regulation of (p)ppGpp synthetase.

Temperature sensitivity of bacteriolysis induced by beta-lactam antibiotics in amino acid-deprived Escherichia coli.

The temperature-sensitive penicillin tolerance response previously reported in amino acid-deprived Escherichia coli (W. Kusser and E. E. Ishiguro, J. Bacteriol. 169:2310-2312, 1987) was not due to the induction of the heat shock response resulting from a temperature upshift and was therefore unrelated to the findings of another report (J. K. Powell and K. D. Young, J. Bacteriol. 173:4021-4026, 1991) indicating a positive correlation between the expression of heat shock proteins and penicillin tolerance. The thermosensitive event occurred in the lysis induction stage.

Occurrence of homologs of the Escherichia coli lytB gene in gram-negative bacterial species.

The Escherichia coli LytB protein regulates the activity of guanosine 3',5'-bispyrophosphate synthetase I (RelA). A Southern blot analysis of chromosomal DNA with the E. coli lytB gene as a probe revealed the presence of lytB homologs in all of the gram-negative bacterial species examined but not in gram-positive species. The lytB homologs from Enterobacter aerogenes and Pseudomonas fluorescens complemented the E. coli lytB44 mutant allele.

Soft X-ray photochemistry beamline at SPring-8.

Design and construction of a soft X-ray beamline at SPring-8 is reported. The beamline utilizes high-quality linearly polarized soft X-rays obtainable from a figure-8 undulator for the study of photophysical and photochemical processes of atoms, molecules and surfaces in the inner-shell excitation region. It consists of two experimental stations, a photochemistry station and a chemical vapour deposition (CVD) station. A high-resolution grating monochromator is installed at the photochemistry station, while the intense undispersed undulator radiation is used at the CVD station. Unique features of the experimental chambers and of the analysis and characterization systems are described along with those of the monochromator.

Comparison of monochromators with a bent parabolic mirror and a varied-spacing grating for the 2.0 GeV high-brilliance synchrotron radiation source (VSX).

A design study of monochromators for a 2.0 GeV electron/positron storage ring for high-brilliance synchrotron radiation in the vacuum ultraviolet (VUV) and the soft X-ray regions is described. Two types of VUV/soft X-ray grazing-incidence monochromators, one with a bent parabolic mirror and the other with a varied-spacing grating, are designed. Without any slope error, the expected resolving power of the former is much higher, but the latter is less affected by slope errors of the optical elements.

Ampicillin-induced bacteriolysis of Escherichia coli is not affected by reduction in levels of anionic phospholipids.

Anionic phospholipids have been shown to interact with both membrane-associated proteins and integral membrane proteins. The objective of this work was to determine whether bacteriolysis induced by treatment with ampicillin was influenced by the levels of anionic membrane phospholipids in Escherichia coli strain HDL11. The pgsA gene, encoding phosphatidylglycerophosphate synthase, in HDL11 is under the control of lacOP, and the levels of anionic membrane phospholipids are consequently dependent on IPTG. The results indicate that limiting the amounts of phosphatidylglycerol and cardiolipin did not affect the lysis process in both growing and nongrowing bacteria.

Cloning and characterization of the lytB gene of Campylobacter jejuni.

LytB of Escherichia coli is an essential gene involved in penicillin tolerance and the stringent response. The lytB gene of Campylobacter jejuni was cloned and characterized. It could complement a temperature-sensitive E. coli lytB mutant. The C. jejuni lytB gene encodes a protein of 277 amino acids that has 34, 36 and 40% amino acid identity with the LytB proteins of E. coli, Haemophilus influenzae, and Synechocystis sp. PCC6803, respectively. The lytB gene is situated between the aroA gene and a gene that encodes ribosomal protein S1.

Dependence of peptidoglycan metabolism on phospholipid synthesis during growth of Escherichia coli.

The role of phospholipid synthesis in peptidoglycan metabolism during growth of Escherichia coli was determined. The inhibition of phospholipid synthesis, achieved by inhibiting fatty acid synthesis with cerulenin or by glycerol deprivation of gpsA mutant strains, resulted in the concomitant inhibition of peptidoglycan synthesis. These effects on peptidoglycan synthesis were relatively specific in that the treatments did not cause a general inhibition of macromolecular synthesis. Furthermore, the inhibition of phospholipid synthesis also resulted in the rapid development of penicillin tolerance. It was unlikely that penicillin tolerance in these cases were simply due to the inhibition of growth caused by cerulenin treatment or glycerol deprivation because treatments with more effective growth inhibitors, e.g. chloramphenicol or norfloxacin, did not confer penicillin tolerance. Penicillin tolerance was shown to be a direct consequence of the inhibition of phospholipid synthesis and not due to the possible accumulation of guanosine-3',5'-bispyrophosphate (ppGpp), the starvation stress signal molecule known to be responsible for the development of penicillin tolerance in amino-acid-deprived bacteria. Therefore, peptidoglycan metabolism is coupled to phospholipid synthesis during growth of E. coli, and this may represent an important means to ensure the coordination of cell envelope synthesis in growing bacteria.

Effects of inhibitors of protein synthesis on lysis of Escherichia coli induced by beta-lactam antibiotics.

The role of protein synthesis in ampicillin-induced lysis of Escherichia coli was investigated. The inhibition of protein synthesis through amino acid deprivation resulted in the rapid development of ampicillin tolerance as a consequence of the stringent response, as previously reported. In contrast, inhibition of protein synthesis by use of ribosome inhibitors such as chloramphenicol did not readily confer ampicillin tolerance and, in fact, promoted the development of both stages of the ampicillin-induced lysis process, i.e., (i) an ampicillin-dependent stage which apparently involves the interaction of penicillin-binding proteins with ampicillin and (ii) an ampicillin-independent stage which may represent the events leading to the deregulation of peptidoglycan hydrolase activity. We propose that lysis was facilitated when protein synthesis was inhibited because the production of new penicillin-binding proteins to replace those which were ampicillin inhibited was prevented under these conditions.

Inhibition of peptidoglycan hydrolase activity in vivo and in vitro by energy uncouplers in Escherichia coli.

The effects of energy uncouplers on in vivo and in vitro peptidoglycan hydrolase activities in Escherichia coli were determined. Sodium azide, potassium cyanide, and carbonyl cyanide m-chlorophenylhydrazone all inhibited ampicillin-induced lysis of exponential phase cultures, even when they were added to lysis-committed cultures. These energy uncouplers also inhibited the solubilization of radiolabeled peptidoglycan by bacterial suspensions that had been treated with 5% trichloroacetic acid by the method of Hartmann et al.3 to activate the peptidoglycan hydrolases. Therefore, the in vivo and in vitro activities of peptidoglycan hydrolases in E. coli are dependent on membrane energization.

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.

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.

Temperature-sensitive mutation in lytF, a new gene involved in autolysis of Escherichia coli.

A temperature-sensitive mutation in a new Escherichia coli gene, located at 62.5 min on the linkage map and designated lytF, resulted in bacteriolysis at the restrictive temperature. Temperature sensitivity and lytF-mediated lysis were simultaneously suppressed by either of two previously described unlinked mutations designated smhA1 and smhB1. The smhA1 and smhB1 alleles were originally isolated as specific extragenic suppressors of temperature-sensitive mutations in three other genes known as murH (99 min), lytD (13 min) and lytE (25 min) which conferred lysis phenotypes indistinguishable from that of the lytF mutation. The murH, lytD and lytE genes have been proposed to be related on the bases of phenotypic similarities and the specificities of their extragenic suppressors. It is now further proposed that lytF belongs to this group. The isolation of new alleles of smhA and smhB as extragenic suppressors of lytF further supports this proposal.

Identification of the Escherichia coli lytB gene, which is involved in penicillin tolerance and control of the stringent response.

The Escherichia coli lytB gene, which is involved in penicillin tolerance and control of the stringent response, was identified as a previously described open reading frame designated orf316 located in the ileS-lsp operon (0.4 min on the linkage map).

Two new genes (smhA and lytE) apparently functionally related to the murH gene of Escherichia coli.

The murH mutant of Escherichia coli exhibits temperature-sensitive growth and lysis at the restrictive temperature. Temperature-resistant derivatives of the mutant occurred at a frequency of about 3 X 10(-6). All of the seven independent isolates examined were shown to be pseudorevertants carrying extragenic suppressors of murH, which mapped at 24.5 min on the linkage map. One allele, apparently representing a new locus, designated smhA, was characterized further. The smhA mutation by itself conferred no recognizable phenotype. However, smhA suppressed the temperature-sensitive lysis phenotype of the murH mutant. The smhA mutant acquired a spontaneous mutation in another new gene, designated lytE, which was mapped at 25 min. The lytE mutation by itself conferred a temperature-sensitive lysis phenotype indistinguishable from that of the murH mutant. The lytE mutation was suppressed by smhA as well as by another suppressor of murH designated smhB. The suppressor activity of smhA was apparently relatively specific in that smhA failed to prevent lysis caused by either mutational or antibiotic-induced blocks in peptidoglycan synthesis. The possibility that the smhA and lytE genes are functionally related to murH is considered.

Complementation of the lytD1 mutation of Escherichia coli by either the cI or cro gene of bacteriophage lambda.

The lytD1 mutant of Escherichia coli exhibits temperature-sensitive growth which is attributed to cellular autolysis at the restrictive temperature. Either of two cloned phage lambda genes, identified as cI and cro, suppressed the lytD1(Ts) lysis phenotype, suggesting that lytD encodes a DNA-binding protein with a DNA-binding specificity similar to that of CI and Cro. LytD may be a repressor of a gene(s) involved in cellular autolysis.

Two new mutant loci (smhB and lytD) in Escherichia coli which confer temperature-sensitive growth and lysis phenotypes.

A temperature-sensitive mutation in the murH gene of Escherichia coli confers a lysis phenotype at the restrictive temperature. An extragenic suppressor of murH apparently representing a new locus at 12.5 min on the linkage map and designated smhB is described. The smhB mutation by itself also conferred a temperature-sensitive lysis phenotype. A mutation in another new locus designated lytD which arose spontaneously in the smhB mutant was mapped close to smhB at 12.7 min on the linkage map. The lytD mutation by itself conferred a temperature-sensitive lysis phenotype indistinguishable from that of the murH mutant. Thus, the suppression of lysis in the smhB murH and the smhB lytD double mutants suggests a mechanism involving the reciprocal suppression of the two individual lysis-causing mutant alleles. The suppressor activity of smhB was apparently relatively specific in that smhB failed to prevent lysis induced by either mutational (murE or murF) or antibiotic-induced blocks in peptidoglycan synthesis. This suggests that murH, smhB, and lytD may be functionally related.