Streptomycin bactericidal action is dependent on polyamine endogenous levels in E. coli.

E. coli polyamine-supplemented and depleted cultures showed an important difference in survival to streptomycin; the bactericidal effect of the antibiotic was remarkably higher in cells with normal levels of polyamines. Similar results were observed with kanamycin. Analysis of the polyamine-containing cells pulse-labelled with 35S-methionine during streptomycin action indicated that the amounts of newly-synthesized peptides in various subcellular fractions was different from the amounts formed in the untreated controls; the most dramatic change was found in the residual particulate fraction where the antibiotic treatment caused a 3-fold increase of radioactive proteins. On the contrary, equivalent amounts of labelled peptides were detected in the different fractions prepared from polyamine-depleted bacteria incubated with or without antibiotic. In this case the corresponding residual fraction was only slightly increased. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the different fractions showed some changes elicited by streptomycin in the protein patterns of polyamine-containing bacteria, especially in the residual fractions. The electrophoretic profile corresponding to deprived cells was very similar in all cases. The role of polyamines in the conformation of the outer membrane and in the correct assembly of ribosomes is discussed on account of the enhancing effect of these polycations on the bactericidal action of streptomycin.

In vitro studies on ppGpp synthetase I from polyamine-starved and unstarved Escherichia coli.

We have studied the in vitro formation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) using a partially purified ppGpp synthetase I (PSI) from Escherichia coli BGA8, a polyamine auxotrophic strain. A comparison of the enzyme obtained from polyamine-supplemented or deprived bacteria showed similar requirements for the reaction, Mg+2 optimum levels and sparing effect of spermidine. No differences in the inhibitory effects of tetracycline, puromycin and fusidic acid were detected either. However, a modified subcellular distribution, as well as a larger specific activity and a larger stimulation by streptomycin was observed when PSI was prepared from polyamine-depleted bacteria. The role of ribosome assembly and subunit distribution on the altered properties of the enzyme are discussed.

A role for polyamines in the control of ppGpp levels in Escherichia coli.

As an approach to understand the involvement of polyamines in the variation of intracellular guanosine 5'-diphosphate 3'-diphosphate (ppGpp) levels, kinetic studies with several polyamine-requiring relA and spoT Escherichia coli mutants have been carried out. The accumulation and turnover of the nucleotide have been followed under conditions of aminoacid depletion or energy source starvation. The results obtained strongly suggest an important role of the polycations mainly in the degradation of ppGpp, and also in its synthesis mediated by ppGpp synthetase I (PSI).

Polyamines and cell wall organization in Saccharomyces cerevisiae.

Cells of Saccharomyces cerevisiae 179-5, an ornithine decarboxylase mutant (spe-1), showed several ultrastructural abnormalities when cultivated in the absence of polyamines. Besides the appearance of microvacuole-like spaces in the cytoplasm and of deformed nuclei, the most important alterations seemed to be located in the cell wall, which was thicker and of heterogeneous texture, and in the cell membrane, of irregular contour. These modifications could not be evoked by general stress conditions elicited by lack of nutrients. The relative levels of cell wall polysaccharides were altered in polyamine-deprived organisms, giving an envelope with increased mannan and decreased glucan content; this cell wall was incompletely attacked by the lytic enzyme zymolyase. Polyamine depletion led also to some abnormalities in the budding pattern. The above observations suggest the involvement of polyamines in the correct structure and organization of the yeast cell.

Effects of DNA gyrase inhibitors in a polyamine-auxotrophic strain of Escherichia coli.

The polyamine content of the Escherichia coli polyamine-auxotrophic strain BGA 8 seemed to influence the effects of nalidixic acid, an antibiotic acting on subunit A of DNA gyrase. The growth rate was more affected under conditions of putrescine depletion and the inhibition could be partially relieved if the polycation was added back to the culture. DNA synthesis was likewise more sensitive to nalidixic acid in cultures grown without polyamine. The expression of some proteins characteristic of the heat-shock response, evoked by the antibiotic, showed a different persistence according to the presence or absence of polyamines. Novobiocin, acting on subunit B of gyrase, also promoted a differential effect depending on the polyamine content, but in this case putrescine-supplemented cells were more sensitive. The described findings suggest a role of polyamines in all the reactions carried out by gyrase, perhaps due to the influence of the polycations on the state of DNA aggregation.

Altered heat-shock response in polyamine-depleted bacteria.

A polyamine-auxotrophic mutant of E. coli was cultivated in the presence or absence of putrescine and submitted to heat shock over 3 different ranges of temperature. In all cases, protein synthetic capacity measured in comparison to that of cultures at the preshift temperature was much higher in polyamine-depleted bacteria under thermic stress. Addition of putrescine only before the shift-up was able to restore gradually normal control of the relative protein synthetic capacity.

Putrescine distribution in Escherichia coli studied in vivo by 13C nuclear magnetic resonance.

In order to study the intracellular polyamine distribution in Escherichia coli, 13C-NMR spectra of [1,4-13C]putrescine were obtained after addition of the latter to intact bacteria. The 13C-enriched methylene signal underwent line broadening. When the cells were centrifuged after 90 min the cell-bound putrescine peak had a linewidth of 23 Hz, while the supernatant liquid showed an unbound putrescine signal with a linewidth smaller than 1 Hz. By using 13C-enriched internal standards it could be shown that the linewidening was not due to the heterogeneity of the medium or to an in vivo paramagnetic effect. Cell-bound putrescine was liberated by addition of trichloroacetic acid and was therefore non-covalently linked to macromolecular cell structures. Cell-bound [13C]putrescine could be displaced by addition of an excess of [12C]putrescine. When samples of membranes, soluble protein, DNA, tRNA and ribosomes from E. coli were incubated with [1,4-13C]putrescine, strong binding was detected only in the ribosomal and membrane fractions. The ribosome-putrescine complex showed properties similar to those determined with the intact cells. By measuring the nuclear Overhauser enhancements eta, it was possible to estimate that only about 50% of the polyamine was linked to the macromolecules. Determination of the T1 values of free and ribosomal-bound putrescine allowed the calculation of a correlation time, tau c = 4 X 10(-7) s for the latter. T1 and tau c values found for the ribosome-putrescine complex were those expected for a motional regime of slowly tumbling molecules.

Polyamine regulation of stringent control in a polyamine-auxotrophic strain of Escherichia coli.

Escherichia coli BGA8 , a mutant unable to synthesize putrescine, behaves as stringent or relaxed according to the presence or absence of polyamine, respectively, in the culture medium. The relaxed synthesis of RNA can be reverted back to stringent by addition of putrescine or spermidine. The stringent response depends on the concentration of the polyamine in the culture medium. The formation of guanosine 3'-diphosphate 5'-diphosphate elicited by amino acid starvation is stimulated at least 40-fold in putrescine-supplemented bacteria and only about 2-fold in putrescine-depleted cells.

Differential effect of alpha-difluoromethyl-ornithine on the proliferation of Balb 3T3 and chemically transformed 3T3 cells.

In order to study the effect of polyamine depletion on growth and proliferation of untransformed and chemically transformed cells, alpha-difluoromethyl-ornithine (DFMO) was added to cultures of 3T3 cells and their benzo[a]pyrene derivative BP-3T3. Both types of cells stopped their proliferation after 72 hr of treatment with the inhibitor. When DFMO was removed and cells were cultivated afterwards in fresh medium without the drug, untransformed cells resumed growth after a lag period, whereas transformed cells were unable to proliferate unless exogenous polyamine was added. These alterations showed a strict correlation with intracellular polyamine pools, since after removal of DFMO from the culture medium, polyamine concentrations increased to almost normal values in 3T3 cells, but remained at low levels or decreased even more in the transformed cells BP-3T3. The analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled proteins of both cell extracts has indicated that the described control of cell proliferation by intracellular polyamine levels might be related to the synthesis of at least two proteins with molecular weights of about 36,000 and 55,000 daltons.

Polyamines and antibiotic effects on translation.

Protein synthesis was studied in polyamine-auxotrophic mutants of Escherichia coli. The decreased protein synthesizing rate observed both in vivo and in vitro in polyamine-starved bacteria is due to defective 30S ribosomal subparticles which are impaired in the initiation step of translation. Analysis of peptides synthesized in vivo suggests a more extensive misreading by putrescine-depleted bacteria. Cells grown without polyamine show a markedly decreased response to the inhibitory effect and the misreading inducing action of streptomycin in vivo and in vitro. The polyamine-starved bacteria are also less sensitive to other aminoglycoside antibiotics such as neomycin, kanamycin and kasugamycin.

Polyamine requirement for streptomycin action on protein synthesis in bacteria.

The effect of streptomycin on polypeptide synthesis in vivo and in vitro has been investigated using polyamine auxotrophic mutants of Escherichia coli grown in the presence or in the absence of putrescine. We found that streptomycin caused a marked inhibition of protein synthesis in polyamine-supplemented cells whereas bacteria starved for polyamines were less sensitive to the action of the antibiotic. Neomycin, kanamycin and kasugamycin had a behaviour similar to streptomycin while spectinomycin, gentamicin and tetracycline brought about a strong inhibition of protein synthesis both in polyamine-starved and unstarved bacteria. The increase of misreading induced by the addition of streptomycin in vivo was higher in extracts derived from bacteria cultivated in the presence of polyamines. This effect was observed in cell-free systems of streptomycin-sensitive and resistant strains. In contrast, spermidine added in vitro caused an improvement in the accuracy of translocation. Analysis of sodium dodecyl sulphate/polyacrylamide gel electrophoresis of the labelled polypeptides synthesized in vivo seems to indicate that the starvation for polyamine or the presence of streptomycin may lead to premature termination with the appearance of unfinished peptide chains.

Lysine decarboxylase mutants of Escherichia coli: evidence for two enzyme forms.

Mutants with reduced lysine decarboxylase activity were isolated from an Escherichia coli polyamine auxotroph. These mutants could not produce induced enzyme, showing only a very low level of an apparently constitutive form. Both enzyme forms could be demonstrated in the parental strain.

Polyamines and protein synthesis: studies in various polyamine-requiring mutants of Escherichia coli.

Different Escherichia coli mutants auxotrophic for polyamines were studied in order to investigate the relationships among polypeptide synthesis in cell-free systems, ribosomal distribution profiles and endogenous polyamine pools. The in vitro protein synthetic activity and the polyribosomal content were reduced in extracts from putrescine-starved cells of the double mutans MA 255 and MA 261, but not in the arginine-conditional auxotroph DK 6. Putrescine addition to the cultures of all these strains previously starved for polyamines, provoked a shift towards monomers in the equilibrium involving ribosomal particles. Concomitant changes in the intracellular levels of polyamines were observed: putrescine and spermidine increased markedly, and cadaverine disappeared.

Polyamines, equilibrium between ribosomal particles and protein synthesis in bacteria.

When putrescine is added to polyamine starved cultures of an E. coli strain difficient in the biosynthesis of putrescine, the protein synthesis is enhanced almost immediately and the ribosomal pattern changes concomitantly, increasing the ratio 70S monomer/ribosomal subparticles. Studies with cell-free systems derived from polyamine starved or unstarved bacteria show that the translation of synthetic and natural mRNAs is several fold higher in system prepared from cells grown in the presence of polyamines. This effect depends on the ribosomal particles and more specifically on the 30S subunit. The results on association of ribosomal subunits strongly suggest that polyamines are involved in this reaction occurring in vivo.

Ribosomal distribution in a polyamine auxotroph of Escherichia coli.

The distribution of ribosomal particles has been studied in a polyamine-deficient mutant of Escherichia coli by sucrose gradient centrifugation analysis. Lysates from starved cells contained less 70S monomers and 30S subunits but more 50S particles than those prepared from bacteria supplemented with putrescine. The addition of the polyamine to putrescine-depleted cells induced a rapid change of the ribosomal profile. A similar effect could be obtained in vitro by equilibrium dialysis against a polyamine-containing solution. The ribosomal pattern obtained from starved bacteria was specific for polyamine deficiency. We conclude that the changes in ribosomal profiles upon restoration of putrescine levels in previously starved cells denote a shift of the equilibrium between 30S-50S couples and ribosomal subunits.