Polyhydroxyalkanoate degradation is associated with nucleotide accumulation and enhances stress resistance and survival of Pseudomonas oleovorans in natural water microcosms.

Pseudomonas oleovorans GPo1 and its polyhydroxyalkanoic acid (PHA) depolymerization-minus mutant, GPo500 phaZ, residing in natural water microcosms, were utilized to asses the effect of PHA availability on survival and resistance to stress agents. The wild-type strain showed increased survival compared to the PHA depolymerase-minus strain. The appearance of a round cellular shape, characteristic of bacteria growing under starvation conditions, was delayed in the wild type in comparison to the mutant strain. Percent survival at the end of ethanol and heat challenges was always higher in GPo1 than in GPo500. Based on these results and on early experiments (H. Hippe, Arch. Mikrobiol. 56:248-277, 1967) that suggested an association of PHA utilization with respiration and oxidative phosphorylation, we investigated the association between PHA degradation and nucleotide accumulation. ATP and guanosine tetraphosphate (ppGpp) production was analyzed under culture conditions leading to PHA depolymerization. A rise in the ATP and ppGpp levels appeared concomitant with PHA degradation, while this phenomenon was not observed in the mutant strain unable to degrade the polymer. Complementation of the phaZ mutation restored the wild-type phenotype.

Pseudomonas aeruginosa UV-A-induced lethal effect: influence of salts, nutritional stress and pyocyanine.

The presence of NaCl in plating media shows an important protection against the Pseudomonas aeruginosa UV-A-induced lethal effect, contrasting with the known sensitizing action of salts on UV-A-irradiated Escherichia coli cells. MgSO4 exhibits a similar protection, but lower concentrations than for NaCl are needed to achieve the same effect. NaCl protection from lethal effects involves an osmotic mechanism, while MgSO4 could act by a different process. On the other hand, when cells grown in a complete medium are then incubated for 20 min in a synthetic medium and irradiated with UV-A, a very marked protection is obtained. This protection is dependent on protein synthesis, since treatment with tetracycline, during the nutritional stress, blocks its induction. These results offer a new example of cross-protection among different stressing agents. In our experimental conditions, natural phenazines of P. aeruginosa are not present in the cells, ruling out the possibility that these pigments act as photosensitizers. Conversely, pyocyanine (the major phenazine produced by this microorganism) prevents the UV-A killing effect in a concentration-dependent way when present in the irradiation media. Finally, UV-A irradiation induces, as in E. coli, the accumulation of guanosine tetraphosphate and guanosine pentaphosphate, although the physiological meaning of this finding has yet to be determined.

Ultraviolet-B lethal damage on Pseudomonas aeruginosa.

Pseudomonas aeruginosa has shown an increased sensitivity compared with that of Escherichia coli and Enterobacter cloacae, when they were exposed to 0.4 kJ/m2 of ultraviolet-B radiation. The rapid decay in cell viability observed in Pseudomonas aeruginosa after the irradiation was influenced by factors such as culture media and the presence of pyocyanine during the irradiation. The radioinduced lethal damage could be prevented by photoreactivating treatment, indicating that pyrimidine dimer formation was the mechanism causing bacterial death. The results indicate that several environmental conditions may act as protective agents against ultraviolet-B-induced damage.

Lethal effect induced in Pseudomonas aeruginosa Exposed to Ultraviolet-A radiation.

Ultraviolet-A (365 nm, 120 kJ/m2/h) exposure caused cell death in Pseudomonas aeruginosa at doses at which Escherichia coli cell viability was not affected. We have not found that UVA induced growth delay or any other sublethal effect. Irradiated suspensions of P. aeruginosa showed a marked reduction in membrane-bound succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) activities. Succinate-driven respiration and several nutrient transport systems were also inhibited. Whereas SDH and LDH activities were independent of the irradiation conditions, cell viability, respiration and transport systems were protected when irradiation was performed in an N2 atmosphere. A similar protective effect was observed when cells were grown in media containing glycerol or when preirradiation bacterial growth was carried out at 30 degrees C (instead of 37 degrees C). Results suggest that UVA induces a differential damaging effect on several biochemical functions of P. aeruginosa. The UVA- induced photodamage may fall into two categories: indirect damage mediated by oxygen (cell killing and inhibition of respiration and transport systems) and direct damage to SDH and LDH (apparently not oxygen dependent). These enzymes and leucine transport appear not to be involved in the lethal effect described herein because they were altered despite viability-preserving conditions

Peptidoglycan hydrolases in an autolytic mutant of Salmonella typhimurium.

Two peptidoglycan hydrolases were isolated from the autolytic mutant Salmonella typhimurium DA361 (envD). One of them, resistant to penicillin, was found free in the supernatant of partially purified envelopes sedimented by ultracentrifugation, and the other bound to the envelopes proved to be sensitive to the antibiotic. Both were able to hydrolyse in vitro high molecular weight non-specific peptidoglycan isolated from E. coli W7 labelled with [14C]diaminopimelic acid. Similar enzymatic activities were separated also from S. typhimurium DA362 (envD+) a non-lytic isogenic pair of the above and from the wild type strain LT-2. All of the hydrolytic activities reported here were strongly inhibited when DNA was added to the assay systems. The peptidoglycan hydrolases isolated from the autolytic mutant suffered a competitive inhibition while those from the non-lytic strains were apparently inhibited in uncompetitive modal relationship. It is postulated that the inhibitory effect may bear affinity with the preservation of DNA sites of attachment to cell membranes sustaining peptidoglycan structure and functions.

Bacteriostatic action of streptomycin on ribosomally resistant mutants (rpsL) of Salmonella typhimurium.

Incubation of streptomycin-resistant (rpsL) mutants of Salmonella typhimurium in alkaline nutrient medium containing streptomycin brought about an inhibition of cell growth that was readily reversed by removing the antibiotic or neutralizing the medium. Growth inhibition was maximal at pH 8.2 and a streptomycin concentration of 800 micrograms/ml. A similar amount of dihydrostreptomycin had a negligible effect, and 10-times-higher concentrations of this antibiotic were required to reproduce the streptomycin action. Addition of streptomycin (400 micrograms/ml) to rpsL cells in alkaline (pH 8.2) nutrient medium caused inhibition of protein and DNA synthesis and also, but to a lower degree, of RNA synthesis. This effect on macromolecular synthesis was not due to ATP deprivation, since ATP content rose after addition of the antibiotic. At pH 8.2, the rate of entrance of streptomycin increased fourfold with respect to the rate at pH 7.0, leading to a large accumulation of streptomycin into rpsL cells. Uptake of the antibiotic was halted by addition of KCN or chloramphenicol. Equal uptake was obtained with 800 micrograms of dihydrostreptomycin or 400 micrograms of streptomycin per ml, yet the former did not affect cell growth at that concentration. It is concluded that high pH stimulates streptomycin and dihydrostreptomycin uptake by rpsL strains but only streptomycin accumulation causes growth inhibition in cells lacking the high-affinity ribosomal site.