Enzymes involved in the biosynthesis of alginate byPseudomonas aeruginosa

Summary Analysis of the enzymes involved in the biosynthesis of alginic acid by mucoidPseudomonas aeruginosa PAO strain's determined the presence of enzymes required to synthesise GDP-mannuronic acid. Addition of polymannuronic acid to an ammonium sulphate precipitate of a cell free alginate suspension indicated the presence of an enzyme which catalysed the epimerisation of mannuronic acid to guluronic acidafter the polymer had been synthesised. The epimerase was shown to be calcium dependant.Various non-mucoid mutants were also studied. The non-mucoid parental strain PAO 381 also contained the enzymes required for alginate synthesis but they were not expressed. Synthesis of alginic acid led to an increase in the level of these enzymes. In the non-mucoid mutants derived from mucoid parents GDP-mannose dehydrogenase was absent in all strains studied. In some of these strains GDP-mannose pyrophosphorylase was also absent, while in other strains, phosphomannase isomerase was absent or greatly reduced.     

Iridescent material and the effect of iron on its production byPseudomonas aeruginosa

The nutritional conditions controlling iridescence inPseudomonas aeruginosa were studied using synthetic media solidified with agar. Iron and magnesium were growth-limiting factors in media solidified with dialysed agar. Iridescence only occurred on iron-deficient media and was not suppressed by adding Ca, Cu, Mn and Zn to these media. The ultraviolet absorption spectrum of the iridescent material was almost identical to the spectrum of the pyo I substances which are 2-alkyl-4-quinolinols.The amount of material produced was inversely proportional to the iron content of the medium. Small amounts of material were produced by cells grown at levels of iron optimal for growth. Synthesis of 2-alkyl-4-quinolinol may be a normal metabolic process in the iridescent strains ofPseudomonas aeruginosa. It was enhanced by anthranilic acid and tryptophan; kynurenine and kynurenic acid had no effect. The results can be explained if it is assumed that the activity of iron-requiring enzymes catalizing the breakdown of tryptophan is reduced.Even in the presence of anthranilic acid or tryptophan no material was produced by a non-iridescent strain.     

Postantibiotic effects and postantibiotic sub-MIC effects of ciprofloxacin, pefloxacin and amikacin on the biological properties ofSalmonella strains

The postantibiotic effect (PAE) and the postantibiotic sub-MIC effect (PASME) of ciprofloxacin, pefloxacin and amikacin were studied forSalmonella typhimurium andS. enteritidis strains. PAE was induced by 2× and 4×MIC of antibiotics studied for 0.5 h. After PAE and PASME their effect on prophage induction of a lysogenicS. typhimurium strain and on Congo red binding for both strains as a marker of their surface hydrophobicity was examined. The longest PAE was found after treatment with ciprofloxacin, higher values being observed withS. typhimurium. PAEs of pefloxacin and amikacin were much lower, except for the suprainhibitory concentration 4×MIC of amikacin withS. enteritidis (6.9 h). PASMEs of ciprofloxacin did not allow any regrowth of either strain. For other antibiotics the PASME's were different while concentrations of 2×MIC+0.2×MIC and 0.3×MIC, and of 4×MIC+0.1×MIC, 0.2×MIC and 0.3×MIC of amikacin did not allow any regrowth ofS. enteritidis. PAEs of the antibiotics tested did not affect the Congo red binding by bothSalmonella strains, but the PAEs of ciprofloxacin and pefloxacin expressively induced a prophage of lysogenicS. typhimurium strain. We noted the influence of Congo red binding after applying 4×MIC+0.1×MIC, 0.2×MIC and 0.3×MIC of amikacin forS. typhinurium and 2×MIC+0.1×MIC forS. enteritidis.     

Influence of aeration on hydrogen cyanide biosynthesis byPseudomonas aeruginosa

Batch cultures ofPseudomonas aeruginosa were able to produce only low levels of cyanide during logarithmic growth with adequate aeration. The reduction of aeration caused a rapid increase in the ability of such cultures to produce hydrogen cyanide. The immediacy and the magnitude of this response depended on the oxygen level, with a concentration of 4% in the aeration gas giving optimal results. The reestablishment of normal aeration resulted in a cessation of the increase of the culture's cyanogenic capacity. This effect appeared to be a combination of inactivation of the hydrogen cyanide synthase and repression of synthesis of this enzyme.     

Kinetic studies on surfactant production byPseudomonas aeruginosa 44T1

Summary Biosurfactant accumulation occurred in the exponential and stationary phases. Production started when the nitrogen level was very low. Surfactant was produced with a diauxic pattern. Rhamnolipid concentration increased as nitrogen levels increased. Maximum product yield (Y _p/x) 2.9 was detected when C/N ratio was 6.6 and specific rate of product formation (p _q) was calculated. The examination of these kinetics parameters such as product yield and specific rate of product formation should be taken into account to develop a high efficient production process.     

Hemolytic effect of a glycolipid produced byPseudomonas aeruginosa

Summary The toxicity of the glycolipid ofPs. aeruginosa to mice has to be ascribed to the fact that it causes hemolysis by dissolving materials from the cell wall structure of the red blood cells.     

Effect of carbon and nitrogen sources on neutral proteinase production byPseudomonas aeruginosa

A strain ofPseudomonas aeruginosa from soil produced large quantitaties of extracellular neutral proteinase and could utilize several organic substances as carbon and nitrogen sources for enzyme production. The growth media required the presence of a high amount of phosphate when glucose was the carbon source. The intermediates of citric-acid cycle acids supported the proteinase production more than any other carbon sources. However, complex nitrogenous substances supported enzyme production more efficiently. Higher concentration of casamino acids suppressed the proteinase synthesis.     

Effect of the carbon source on biosurfactant production byPseudomonas aeruginosa 44T1

Summary Pseudomonas aeruginosa 44T1 produces rhamnolipids when grown on C₁₂ n-alkane but not with other hydrocarbons tested. Best results were obtained with olive oil as carbon source; a final production of 7.65 g rhamnolipid/l with a production yield of 38.2% was detected.     

Optimization of lipase production byPseudomonas aeruginosa MB 5001 in batch cultivation

Summary The production ofPseudomonas aeruginosa MB 5001 extracellular lipase was optimized by batch cultivation employing shake flasks and 23-L bioreactors. This enzyme efficiently and selectively bioconverts dimethyl 5-(3-(2-(7-chloroquinolin-2-yl)ethyl)phenyl)4,6-dithianonanedioate (diester) to its (S)-ester acid. Process development studies focused on the identification and optimization of the physicochemical parameters required to achieve maximum lipase production. Of the media evaluated, a peptonized milk-based medium was found to support excellent lipase production and stability. Medium composition and process parameters that supported optimal lipase production were different from those supporting maximum biomass formation. Of the parameters investigated, dissolved oxygen tension had the most significant and unexpected impact on lipase production. Elevated lipase production was achieved whenP. aeruginosa MB 5001 was cultivated in a dissolved oxygen limited environment. Overall, these process development studies resulted in a 100% increase in lipase production when compared to the original shake flask process employing skim milk.     

The effect of electrical currents and tobramycin onPseudomonas aeruginosa biofilms

The combined use of antibiotics with low levels of electrical current has been reported to be more effective in controlling biofilms (the bioelectric effect) than antibiotics alone. An electrical colonisation cell was designed to study the effect of antibiotics on biofilms formed on a dialysis membrane away from the electrode surface. To avoid the electrochemical generation of toxic products,Pseudomonas aeruginosa biofilms were formed in minimal salts medium that excluded chloride-containing compounds. Under these conditions, electrical currents of up to 20 mA cm^–2 did not prevent biofilm formation or have any detrimental effect on an established biofilm. Tobramycin alone at concentrations of 10 g ml^–1 did not affect the biofilm, but were significantly enhanced by 9 mA cm^–2. The effect of tobramycin concentrations of 25 g ml^–1 were enhanced by a 15 mA cm^–2 electrical current. In both cases higher levels of electrical current, up to 20 mA cm^–2, did not further enhance the effect of the antibiotic. The possible mechanisms of action of the bioelectric effect have been reported to involve electrophoresis, iontophoresis and electroporesis, thus overcoming the biofilm biomass and cell wall barriers. Our results suggest that other factors may also be important, such as the metabolic activity and growth rate of the bacteria. Such factors may be critical in maximising antibiotic efficacy.     

Degradation of different hydrocarbons and production of biosurfactant byPseudomonas aeruginosa isolated from coastal waters

Summary A bacterium isolated from an oil spillage sample and identified asPseudomonas aeruginosa degraded hexadecane and heptadecane by 47% and 58% with 9% and 12% of total carbon from the respective substrates being liberated as CO₂. With octadecane and nonadecane as substrates, 73% and 60% were biodegraded while 27% and 25% of total carbon was evolved as CO², respectively. Production of biosurfactant by this bacterium was studied using hexadecane 5% (v/v) as substrate. The surface tension of spent culture medium, as well as the supernatant, was 30 mN/m compared to 71 mN/m for water. When the supernatant was mixed with hexadecane (15, v/v) a stable emulsion was formed which deteriorated only by 10% after one month.

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Dégradation de différents hydrocarbures et production de biosurfactants parPseudomonas aeruginosa isolé d'eaux côtières

Résumé Une bactérie, isolée d'un échantillon d'hydrocarbures épandus, et identifiée commePseudomonas aeruginosa, dégrade l'hexaet l'heptadécane à raison respective de 47 et de 58% du carbone total avec 9 et 12% des substrats respectifs libérés sous la forme de CO₂. Avec l'octa- et le nonadécane comme substrats, la biodégradation atteint respectivement 73 et 60% avec 27 et 25% du carbone total transformé en CO₂. On a étudié la production de biosurfactant par cette bactérie en utilisant l'hexadécane à 5% (v/v) comme substrat. La tension superficielle de la liqueur mixte, comme celle de son surnageant atteint 30 mN/m par comparaison à 71 mN/m pour l'eau. Lorsque le surnageant est mélangé à l'hexadécane (15, v/v), on forme une émulsion stable qui ne se détériore que de 10% au bout d'un mois.

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This work was carried out at the Biotechnology Laboratories of the Nuclear Institute for Agriculture & Biology (NIAB), Faisalabad.     

Products of the oxidation ofn-decane byPseudomonas aeruginosa andMycobacterium rhodochrous

Pseudomonas aeruginosa andMycobacterium rhodochrous (both soil isolates) have been grown in a mineral salts medium containing hydrocarbon as the only carbon source. The fermentation products from decane withPseudomonas aeruginosa were 1-, 2-, 3-, and 4- and or 5-decanol and the corresponding ketones (no decanal was found) and decanoic, nonanoic, octanoic and heptanoic acid. This indicates that non-specific first oxidation occurs followed by seission of the decanone at the keto group. The products fromMycobacterium rhodochrous were 1-decanol,n-decanal, decanoic, octanoic, hexanoic, butyric and acetic acids, indicating that initial terminal oxidation is followed by-oxidation.     

In vitro alginate polymerization and the functional role of Alg8 in alginate production by Pseudomonas aeruginosa

An enzymatic in vitro alginate polymerization assay was developed by using 14C-labeled GDP-mannuronic acid as a substrate and subcellular fractions of alginate overproducing Pseudomonas aeruginosa FRD1 as a polymerase source. The highest specific alginate polymerase activity was detected in the envelope fraction, suggesting that cytoplasmic and outer membrane proteins constitute the functional alginate polymerase complex. Accordingly, no alginate polymerase activity was detected using cytoplasmic membrane or outer membrane proteins, respectively. To determine the requirement of Alg8, which has been proposed as catalytic subunit of alginate polymerase, nonpolar isogenic alg8 knockout mutants of alginate-overproducing P. aeruginosa FRD1 and P. aeruginosa PDO300 were constructed, respectively. These mutants were deficient in alginate biosynthesis, and alginate production was restored by introducing only the alg8 gene. Surprisingly, this resulted in significant alginate overproduction of the complemented P. aeruginosa Deltaalg8 mutants compared to nonmutated strains, suggesting that Alg8 is the bottleneck in alginate biosynthesis. (1)H-NMR analysis of alginate isolated from these complemented mutants showed that the degree of acetylation increased from 4.7 to 9.3% and the guluronic acid content was reduced from 38 to 19%. Protein topology prediction indicated that Alg8 is a membrane protein. Fusion protein analysis provided evidence that Alg8 is located in the cytoplasmic membrane with a periplasmic C terminus. Subcellular fractionation suggested that the highest specific PhoA activity of Alg8-PhoA is present in the cytoplasmic membrane. A structural model of Alg8 based on the structure of SpsA from Bacillus subtilis was developed.     

Effects of methanol on the synthesis of aspartame precursor catalysed byPseudomonas aeruginosa elastase

Summary Pseudomonas elastase was found to be efficient in catalysing the reaction betweenN-benzyloxycarbonyl-L-aspartic acid and L-phenylalanine methyl ester producing the aspartame precursor in aqueous and aqueous methanolic solutions. 25% (v/v) methanol was most favourable for the synthesis where about 100% increase in yield was obtained compared to that in aqueous solution.Abbreviations N-cbz-L-Asp N-benzyloxycarbonyl-L-Aspartic acid - L-Phe-OMe L-phenyl alanine methyl ester - HPLC high performance liquid chromatography. All the % of methanol is a volume % in water unless otherwise specified     

The effects of 2,4-dinitrophenol on the oxidation of fatty acids byPseudomonas aeruginosa

Summary The effects of varying concentrations of 2,4-dinitrophenol on the oxidation of a series of saturated, aliphatic fatty acids byPs. aeruginosa were studied. The oxidation of C₄, C₆, C₈, C₁₀, C₁₂, C₁₃, C₁₄ and C₁₆ acids is stimulated by certain concentrations of this inhibitor. However, the concentration of 2,4-dinitrophenol which causes stimulation of oxygen uptake with capric acid does not produce an increase in numbers of the organisms in a medium containing the fatty acid as the sole carbon source. This investigation was supported by a Fellowship from the Anderson Oil and Chemical Company, Inc., Portland, Connecticut.     

Experimental and theoretical studies on the molecular properties of ciprofloxacin,norfloxacin, pefloxacin,sparfloxacin, and gatifloxacin in determining bioavailability

The aim of this investigation is to identify, by in silico and in vitro methods, the molecular determinants, e.g., solubility in an aqueous medium and lipophilic properties, which have an effect on the bioavailability of five selected fluoroquinolones. These properties were estimated by analysis of the electrostatic potential pattern and values of free energy of solvation as well as the partition coefficients of the studied compounds. The study is based on theoretical quantum-chemical methods and a simple experimental shake-flask technique with two immiscible phases, n-octanol and phosphate buffer. The solvation free energy values of compounds in both environments appeared to be negative. The wide range of electrostatic potential from negative to positive demonstrates the presence of dipole–dipole intermolecular interactions, while the high electron density at various sites indicates the possibility of hydrogen bond formation with solvent molecules. High partition coefficient values, obtained by summing the atomic contributions, did not take various correction factors into account and therefore were not accurate. Theoretical partition coefficient values based on more accurate algorithms, which included these correction factors (fragmental methods), yielded more accurate values. Theoretical methods are useful tools for predicting the bioavailability of fluoroquinolones.     

氨溴索对粘液型铜绿假单胞菌生物膜藻酸盐作用的体外研究

目的探讨氨溴索对铜绿假单胞菌临床分离株形成的生物膜（biofilm,BF）主要成分藻酸盐的干预作用,研究其对藻酸盐合成过程中起重要作用的基因表达和合成过程中限速酶活性的影响,以及其对藻酸盐降解的影响。方法建立铜绿似单胞菌临床分离株BF体外模型,培养7d后得到成熟BF。将BF内的细菌振荡下来后,用疏酸-苯酚法检测氨溴索对藻酸盐含量的影响;RT-PCR检测藻酸盐合成过程中重要基因algD、algU、algR和mucA的mRNA表达;分光光度计检测合成过程中限速酶——GDP-甘露糖脱氢酶（guanosine diphospho-D-mannose dehydrogenase,GMD）的活性,并检测藻酸盐的降解情况。结果在氨溴索3．75mg／ml作用下,藻酸盐含量（mg／g）由86．4024±0．8588下降到59．9199±0．5803（F=66．2,P〈0．01）;其合成重要基因algD、algU、algR和mucA的mRNA的表达分别由1．2994±0．0173、1．0488±0．0457、0．9888±0．0267和0．8731±0．0336变化为1．0253±0．0265、0．9594±0．0106、0．8536±0．0179和1．0770±0．0503（F=91．9,41．1,88．4和56,9,P均〈0．05）;其合成限速酶GMD活性由0．0989±0．0055下降到0．0558±0．0016（F=121．2,P〈0．01）;藻酸盐的降解量（△mg／g）由1．4122±0．0073变化为1．4175±0．0019（F=21．81,P〉0．05）。1．875mg／ml氨溴索作用下,有同样的趋势但效应不如高浓度明显。结论氨溴索可以降低铜绿假单胞菌BF藻酸盐的含量,影响藻酸盐合成过程中重要基因algD、algU、algR和mucA的mRNA的表达,降低藻酸盐合成限速酶GMD活性,但对藻酸盐的降解无影响。     

Nitrosative stress inhibits production of the virulence factor alginate in mucoid Pseudomonas aeruginosa

Alginate is a critical virulence factor contributing to the poor clinical prognosis associated with the conversion of Pseudomonas aeruginosa to mucoid phenotypes in cystic fibrosis (CF). An important mechanism of action is its ability to scavenge host innate-immune reactive species. We have previously analyzed the bacterial response to nitrosative stress by S-nitrosoglutathione (GSNO), a physiological NO√ donor with diminished levels in the CF lung. GSNO substantially increased bacterial nitrosative and oxidative defenses and so we hypothesized a similar increase in alginate production would occur. However, in mucoid P. aeruginosa, there was decreased expression of the majority of alginate synthetic genes. This microarray data was confirmed both by RT-PCR and at the functional level by direct measurements of alginate production. Our data suggest that the lowered levels of innate-immune nitrosative mediators (such as GSNO) in the CF lung exacerbate the effects of mucoid P. aeruginosa, by failing to suppress alginate biosynthesis.