Induction of Histidine-Degrading Enzymes in Pseudomonas aeruginosa

Urocanate but not histidine was able to induce formation of histidine-degrading enzymes in a histidine ammonia-lyase-deficient mutant of Pseudomonas aeruginosa. The results, in conjunction with others reported previously, indicate that urocanate, the first intermediate, is the physiological inducer of the pathway.     

Secretion of Elastinolytic Enzymes and Their Propeptides by Pseudomonas aeruginosa

Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. The signal sequence is cleaved off during transport across the inner membrane and, in the periplasm, proelastase is further processed. We demonstrate that the propeptide and the mature elastase are both secreted but that the propeptide is degraded extracellularly. In addition, reduction of the extracellular proteolytic activity led to the accumulation of unprocessed forms of LasA and LasD in the extracellular medium, which shows that these enzymes are secreted in association with their propeptides. Furthermore, a hitherto undefined protein with homology to a Streptomyces griseus aminopeptidase accumulated under these conditions.     

Inactivation of Pseudomonas aeruginosa biofilm by dense phase carbon dioxide

Dense phase carbon dioxide (DPCD) is one of the most promising techniques available to control microorganisms as a non-thermal disinfection method. However, no study on the efficiency of biofilm disinfection using DPCD has been reported. The efficiency of DPCD in inactivating Pseudomonas aeruginosa biofilm, which is known to have high antimicrobial resistance, was thus investigated. P. aeruginosa biofilm, which was not immersed in water but was completely wet, was found to be more effectively inactivated by DPCD treatment, achieving a 6-log reduction within 7 min. The inactivation efficiency increased modestly with increasing pressure and temperature. This study also reports that the water-unimmersed condition is one of the most important operating parameters in achieving efficient biofilm control by DPCD treatment. In addition, observations by confocal laser scanning microscopy revealed that DPCD treatment not only inactivated biofilm cells on the glass coupons but also caused detachment of the biofilm following weakening of its structure as a result of the DPCD treatment; this is an added benefit of DPCD treatment.     

Inactivation of allantoinase from Pseudomonas aeruginosa by a subunit of urease

Cell-free extracts prepared from Pseudomonas aeruginosa cells, cultured in a medium containing allantoin as sole source of carbon, nitrogen and energy and harvested in the stationary phase, contain an enzymicly inactive allantoinase-inhibitor complex. Pure inhibitor was isolated by dissociation of this complex followed by gelfiltration. The inhibitor had a molecular weight of about 5500 daltons. Association between inhibitor and allantoinase was demonstrated by gelfiltration and by polyacrylamide gel-electrophoresis. The inhibitor was unstable in the absence of 1 M urea and the inactivation was accompanied by aggregate formation and appearance of urease activity. The inhibitor was also isolated from cells containing urease but no allantoinase. It was concluded that the inhibitor is a subunit of urease. Inhibitors isolated from P. aeruginosa and P. acidovorans cells were active against both allantoinase from P. aeruginosa and allantoinase from P. acidovorans.     

Inactivation and Phosphorylation of Kanamycin by Drug-resistant Staphylococcus aureus

The cell-free system of clinical isolates of drug-resistant Staphylococcus aureus inactivated kanamycin, and the inactivated product was identified with kanamycin-3′-phosphate, in which the C-3-OH of the 6-amino-6-deoxy-d-glucose moiety of kanamycin was phosphorylated.     

Inactivation of the potent Pseudomonas aeruginosa cytotoxin pyocyanin by airway peroxidases and nitrite

Pyocyanin (1-hydroxy-N-methylphenazine, PCN) is a cytotoxic pigment and virulence factor secreted by the human bacterial pathogen, Pseudomonas aeruginosa. Here, we report that exposure of PCN to airway peroxidases, hydrogen peroxide (H(2)O(2)), and NaNO(2) generates unique mononitrated PCN metabolites (N-PCN) as revealed by HPLC/mass spectrometry analyses. N-PCN, in contrast to PCN, was devoid of antibiotic activity and failed to kill Escherichia coli and Staphylococcus aureus. Furthermore, in contrast to PCN, intratracheal instillation of N-PCN into murine lungs failed to induce a significant inflammatory response. Surprisingly, at a pH of ～7, N-PCN was more reactive than PCN with respect to NADH oxidation but resulted in a similar magnitude of superoxide production as detected by electron paramagnetic resonance and spin trapping experiments. When incubated with Escherichia coli or lung A549 cells, PCN and N-PCN both led to superoxide formation, but lesser amounts were detected with N-PCN. Our results demonstrate that PCN that has been nitrated by peroxidase/H(2)O(2)/NO(2)(-) systems possesses less cytotoxic/proinflammatory activity than native PCN. Yield of N-PCN was decreased by the presence of the competing physiological peroxidase substrates (thiocyonate) SCN(-) (myeloperoxidase, MPO, and lactoperoxidase, LPO) and Cl(-) (MPO), which with Cl(-) yielded chlorinated PCNs. These reaction products also showed decreased proinflammatory ability when instilled into the lungs of mice. These observations add important insights into the complexity of the pathogenesis of lung injury associated with Pseudomonas aeruginosa infections and provide additional rationale for exploring the efficacy of NO(2)(-) in the therapy of chronic Pseudomonas aeruginosa airway infection in cystic fibrosis.     

Inactivation of Pseudomonas aeruginosa PA01 biofilms by hyperthermia using superparamagnetic nanoparticles

The primary goal of this study was to develop a new strategy to inactivate bacterial biofilms using the thermal stress derived from superparamagnetic iron oxide nanoparticles (SPIONs) in an alternating current (AC) magnetic field. A large number of studies have examined the inactivation of bacterial biofilms using antimicrobial agents; however, there have been no attempts to inactivate biofilms by hyperthermia using SPIONs. In this study, a SPION solution was added to Pseudomonas aeruginosa (P. aeruginosa) PA01 biofilm, and heat was generated by placing the nanoparticle-containing biofilm in an AC magnetic field. The heating temperature was dependent on the concentration of the added SPION solution. More than 4 log inactivation of the PA01 biofilm was obtained using a 60 mg mL⁻¹ SPION solution in 8 min, and this resulted in a dramatic disintegration of the bacterial cell membrane in the biofilm. This inactivation was largely due to the thermal effect. Local heating of a specific area is also possible using this method, and the heating temperature can be easily adjusted by controlling the concentration of the SPION solution. Therefore, hyperthermia using magnetic nanoparticles holds promise as an effective tool for inactivating the bacterial biofilm.     

Subinhibitory Concentration of Kanamycin Induces the Pseudomonas aeruginosa type VI Secretion System

Pseudomonas aeruginosa is a Gram-negative bacterium found in natural environments including plants, soils and warm moist surfaces. This organism is also in the top ten of nosocomial pathogens, and prevalent in cystic fibrosis (CF) lung infections. The ability of P. aeruginosa to colonize a wide variety of environments in a lasting manner is associated with the formation of a resistant biofilm and the capacity to efficiently outcompete other microorganisms. Here we demonstrate that sub-inhibitory concentration of kanamycin not only induces biofilm formation but also induces expression of the type VI secretion genes in the H1-T6SS cluster. The H1-T6SS is known for its role in toxin production and bacterial competition. We show that the antibiotic induction of the H1-T6SS only occurs when a functional Gac/Rsm pathway is present. These observations may contribute to understand how P. aeruginosa responds to antibiotic producing competitors. It also suggests that improper antibiotic therapy may enhance P. aeruginosa colonization, including in the airways of CF patients.     

Enzymes Lytic against Pseudomonas aeruginosa Produced by Bacillus subtilis YT-25

A bacterium YT–25 which produces enzymes lytic against Pseudomonas aeruginosa was isolated from soil and it was identified as Bacillus subtilis.

A₁-enzyme, A₂-enzyme, B-enzyme and NLF (Native Cell-Lytic Factor) which contribute the lysis of P. aeruginosa were purified from the culture filtrate of strain YT–25.

Purified A₁-enzyme, A₂-enzyme and B-enzyme individually lysed the vegetative cells of P. aeruginosa in the presence of NLF.

NLF is a low molecular basic peptide and seemed to alter the sufrace structure of P. aeruginosa.

B-enzyme hydrolyzed the peptidoglycan purified from P. aeruginosa to release the reducing groups, but A₁-enzyme and A₂-enzyme released neither reducing groups nor free amino groups from the peptidoglycan.     

等离子体射流灭活液体中铜绿假单胞菌的研究

【目的】测定等离子射流对铜绿假单胞菌(Pseudomonas aeruginosa)的灭活效果,探究低温等离子体射流的杀菌机理。【方法】采用平板计数法测定等离子体射流的杀菌效果,荧光显微镜、透射电镜观察等离子体作用后菌体结构的变化,蛋白浓度测定和SDS-PAGE电泳检测菌液上清液中可溶性蛋白的泄漏量。【结果】等离子体射流处理铜绿假单胞菌菌液5 min,杀灭率可达到99.9%以上。透射电镜观察可见细菌菌体结构发生改变,细胞壁、细胞膜损伤破裂,细胞内容物泄露。进一步对处理铜绿假单胞菌上清液中的蛋白质含量变化进行检测,结果显示随着处理时间的增加,上清液中蛋白质含量持续增加,在2 min时达到最大值。【结论】等离子体射流可以通过破坏细胞结构造成细胞质泄露,使其丧失正常的细胞功能,从而达到快速有效地杀灭铜绿假单胞菌的效果。     

Coresistance to Neomycin and Kanamycin by Mutations in an Escherichia coli Locus that Affects Ribosomes

Mutant strains resistant to neomycin or to kanamycin sulfate were isolated from Escherichia coli K-12. Nine mutants were analyzed; all were resistant to both antibiotics (about 150 and 100 mug/ml, respectively), and were designated nek. In the mutant strains, the ribosomes are changed from those of the parental strain; for when they were used in assays for polypeptide formation directed by polyadenylic acid or polycytidylic acid, coding fidelity in presence of the drugs was increased and inhibition of synthesis by the drugs was lessened. Mating experiments and transduction tests showed that all of the nine nek mutants are either closely linked or allelic, and the nek locus is closely linked to two genes-str (streptomycin) and spc (spectinomycin)-known to affect the 30S ribosome. The two nek mutants tested were recessive to the sensitive, wild-type allele. When the nek mutants were compared to the parental strain, pleiotropic effects of the nek mutations were observed. Resistance to low levels of streptomycin and spectinomycin was increased, whereas resistance to chloramphenicol was decreased. Also, the mutants were less able to adapt to high concentrations of lincomycin, and could no longer show phenotypic suppression of an arginine requirement by neomycin or kanamycin. Such pleiotropic effects are suggested to be the rule for mutations in genes that participate in the biosynthesis of a cellular organelle.     

Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli

R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains. Expression in P. putida was similar to that in P. aeruginosa and was repressed by exogenous arginine. Expression in E. coli was 2 to 4% of that in P. aeruginosa. Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E. coli in this respect.     

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.     

Chelator-Induced Dispersal and Killing of Pseudomonas aeruginosa Cells in a Biofilm

Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacteria in biofilms are more resistant to the immune system and to antibiotics than their free-living planktonic counterparts. Thus, biofilm-related infections are persistent and often show recurrent symptoms. The metal chelator EDTA is known to have activity against biofilms of gram-positive bacteria such as Staphylococcus aureus. EDTA can also kill planktonic cells of Proteobacteria like Pseudomonas aeruginosa. In this study we demonstrate that EDTA is a potent P. aeruginosa biofilm disrupter. In Tris buffer, EDTA treatment of P. aeruginosa biofilms results in 1,000-fold greater killing than treatment with the P. aeruginosa antibiotic gentamicin. Furthermore, a combination of EDTA and gentamicin results in complete killing of biofilm cells. P. aeruginosa biofilms can form structured mushroom-like entities when grown under flow on a glass surface. Time lapse confocal scanning laser microscopy shows that EDTA causes a dispersal of P. aeruginosa cells from biofilms and killing of biofilm cells within the mushroom-like structures. An examination of the influence of several divalent cations on the antibiofilm activity of EDTA indicates that magnesium, calcium, and iron protect P. aeruginosa biofilms against EDTA treatment. Our results are consistent with a mechanism whereby EDTA causes detachment and killing of biofilm cells.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Surface Structure of Intact Cells and Spheroplasts of Pseudomonas aeruginosa

This report describes the ultrastructural features of Pseudomonas aeruginosa after freeze-etching of intact cells and enzymatically prepared spheroplasts. Freeze-etching of intact cells revealed two convex layers of the cell wall and particles within the hydrophobic interior of the cell membrane. Areas of the membrane free of particles were sometimes elevated in the form of rather large dome-shaped structures. Spheroplasts were formed from intact cells by the addition of trypsin to a reaction mixture of lysozyme and ethylenediaminetetraacetic acid. Spheroplasts contained the outer lipoid layer of the cell wall. It was possible to observe this cell wall layer in freeze-etch preparations of spheroplasts. The spheroplast membrane like that of intact cells was cleaved along a central plane to expose particles and particle-free areas.     

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans.

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.