Regulation of enzyme synthesis in the arginine deiminase pathway of Pseudomonas aeruginosa.

The three enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa strain PAO were induced strongly (50- to 100-fold) by a shift from aerobic growth conditions to very low oxygen tension. Arginine in the culture medium was not essential for induction, but increased the maximum enzyme levels twofold. The induction of the three enzymes arginine deiminase (EC 3.5.3.6), catabolic ornithine carbamoyltransferase (EC 2.1.3.3), and carbamate kinase (EC 2.7.2.3) appeared to be coordinate. Catabolic ornithine carbamoyltransferase was studied in most detail. Nitrate and nitrite, which can replace oxygen as terminal electron acceptors in P. aeruginosa, partially prevented enzyme induction by low oxygen tension in the wild-type strain, but not in nar (nitrate reductase-negative) mutants. Glucose was found to exert catabolite repression of the deiminase pathway. Generally, conditions of stress, such as depletion of the carbon and energy source or the phosphate source, resulted in induced synthesis of catabolic ornithine carbamoyltransferase. The induction of the deiminase pathway is thought to mobilize intra- and extracellular reserves of arginine, which is used as a source of adenosine 5'-triphosphate in the absence of respiration.     

Monitoring microaerobic denitrification of Pseudomonas aeruginosa by online NAD(P)H fluorescence

Defined as the transition conditions in which the organism(s) performs simultaneous aerobic and anaerobic respiration or fermentation, microaerobic conditions are commonly present in the nature. Microaerobic metabolism of microorganisms is however poorly characterized. Being extremely sensitive to the change in cellular electron-accepting mechanisms, NAD(P)H fluorescence provides a useful ways for online monitoring of microaerobic metabolism. Its application to studies of microbial nitrate respiration and particularly, denitrification of Pseudomonas aeruginosa is reviewed here, centering on four topics: (1) online monitoring of anaerobic nitrate respiration by NAD(P)H fluorescence, (2) effects of denitrification on P. aeruginosa phenotypes, (3) microaerobic denitrification of P. aeruginosa in continuous culture, and (4) correlation between NAD(P)H fluorescence and denitrification-to-respiration ratio. Online NAD(P)H fluorescence is shown to sensitively detect the changes of cellular metabolism. For example, it revealed the intermediate nitrite accumulation in C-limited Escherichia coli performing anaerobic nitrate respiration via dissimilative ammonification, by exhibiting two-stage profiles with intriguing fluorescence oscillation. When applied to continuous culture studies of P. aeruginosa (ATCC 9027), the online fluorescence helped to identify that the bacterium conducted denitrification even at DO > 1 mg/l. In addition, the fluorescence profile showed a unique correlation with the fraction of electrons accepted by denitrification (out of all the electrons accepted by aerobic and anaerobic respiration). The applicability of online NAD(P)H fluorescence in monitoring and quantitatively describing the sensitive microaerobic state of microorganisms is clearly demonstrated.     

Isolation of an iron-binding compound from Pseudomonas aeruginosa.

An iron-binding compound was isolated from ethyl acetate extracts of culture supernatant fluids of Pseudomonas aeruginosa and was purified by successive paper and thin-layer chromatographic procedures. The purified compound was characterized by UV, visible, infrared, and fluorescence spectroscopy. The compound possesses phenolic characteristics, with little or no similarity to dihydroxybenzoates and no indication of a hydroxamate group. P. aeruginosa synthesized the compound during active growth in culture media containing less than 5 X 10(-6) M added FeCl3. When added to iron-poor cultures of P. aeruginosa, the compound promoted the growth of the bacterium and also reversed growth inhibition by the iron chelator ethylenediamine-di-(o-hydroxyphenylacetic acid).     

Specific oxygen uptake rate variations during batch fermentation of Bacillus thuringiensis subspecies kurstaki HD-1

The specific oxygen uptake rate (q(O)2, respiration rate) of Bacillus thuringiensis subsp. kurstaki HD-1 was very high at inoculation and was found to decrease essentially monotonically throughout both vegetative growth phase and transition phase under different batch culture conditions. Average q(O)2 values decreased from 8-10 mmol/g h at 1 h after inoculation to less than 2 mmol/g h by the time growth ended. The results are shown to be consistent with the few previous reports on q(O)2 in B. thuringiensis in the literature but also novel in that this pattern of monotonic decline has not been described previously. Both pH control and EDTA in low concentration shortened the vegetative growth phase and reduced the 10 h biomass concentration. Using plots of q(O)2 versus specific growth rate, mu, biomass yield based on the oxygen used for growth, was calculated for transition phase to be 0.041-0.047 g/mmol, consistent with literature values. The same plot also showed that the presence of EDTA resulted in an atypical q(O)2-mu trajectory and apparently much higher biomass yield from the oxygen consumed.     

Anti-infective properties of Lactobacillus fermentum against Staphylococcus aureus and Pseudomonas aeruginosa

Surgical wounds and implant-associated Staphylococcus aureus and Pseudomonas aeruginosa infections are often difficult to treat because of limited susceptibility of several of these strains to conventional antibiotics. As a result, there is a constant need for new alternative drugs. The aim of this study was to investigate the antimicrobial properties of Lactobacillus fermentum, a probiotic bacterium, which we have isolated from colonic biopsies. The inhibition of S. aureus and P. aeruginosa growth was evaluated by coincubating with L. fermentum strains. Growth inhibition was tested for several of their clinical isolates using agar well diffusion assays. For biofilm assay S. aureus and P. aeruginosa were grown on the glass slides and in 96-well plates in presence of 2.5 μg/ml culture filtrate of L. fermentum. Biofilms were photographed using confocal microscope or stained with 0.1% crystal violet. Reduction in the cytotoxicity of S. aureus and P. aeruginosa was observed in presence of 2.5 μg/ml L. fermentum-spent media. Using in vitroexperiments, we showed that L. fermentum-secreted compound(s) inhibits the growth, cytotoxicity and biofilm formation of several S. aureus and P. aeruginosa strains. Compound(s) present in the culture supernatant of L. fermentum may have promising applications in treating hospital-acquired infections.     

Use of artificial sputum medium to test antibiotic efficacy against Pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests. Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions. An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a > 128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions. The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods. Several in vitro models have been used previously to study P. aeruginosa biofilms. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung. In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa and affect antibiotic susceptibility. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.     

Autoradiographic study of the localization and evolution of growth zones in bacterial colonies.

Incorporation of [3H]leucine in the bacteria of 18 to 48 h-old colonies of Pseudomonas aeruginosa, Pseudomonas putida, Bacillus thuringiensis, Staphylococcus aureus and Escherichia coli enabled the localization of bacterial multiplication sites by means of autoradiography of sagittal sections. In colonies where fast diameter expansion occurred, all the bacteria from the peripheral corona contributed to peripheral growth; in colonies where the expansion was slower, the growth rate of the bacteria in this region was heterogeneous. Besides this peripheral growth, a central region of bacterial multiplication was always found, but with variable localization and extension. In aerobic species, such as P. aeruginosa and P. putida, the central growth site was limited to the zone of oxygen penetration into the bacterial mass. However, in facultatively anaerobic species, bacterial multiplication dependend on nutrient supply. For 48 h-old colonies of S. aureus, a more complex localization of growth seemed to be affected simultaneously by nutrient penetration and accumulation of toxic substances.     

不同碳源对悬浮培养玫瑰茄细胞呼吸强度及产生辅酶Q的影响

以蔗糖、葡萄糖、可溶性淀粉为３种不同碳源,以Ｂ５培养基为基础,研究在不同碳源情况下,悬浮培养玫瑰茄细胞的生长,以及培养过程中细胞的氧消耗速率,同时也考察了在整个细胞生长过程中,辅酶Ｑ的含量变化。结果表明,蔗糖和葡萄糖能有效支持玫瑰茄细胞的生长,其细胞活力也高,表现在呼吸强度较高;可溶性淀粉不能被植物细胞利用,不仅表现在细胞生物量低,而且细胞活力差,呼吸水平也较低     

Variation of microcystin content of microcystis aeruginosa relative to medium N:P ratio and growth stage

Changes in the microcystin content of Microcystis aeruginosa UTEX 2388 were investigated at several N:P ratios of the medium and various growth stages. Under the P-fixed condition, the microcystin content of the cells changed with different medium N:P ratios, with the highest at 2748 microg g-1 at a N:P ratio of 16 after incubation for 7 d. The microcystin content of M. aeruginosa exhibited a high correlation with the total N content regardless of an N-fixed or P-fixed culture. When the N:P ratio of the medium was fixed to 16 : 1, the microcystin content of M. aeruginosa at various growth stages was highest at 2191 microg g-1 after an incubation of 4 d and the chlorophyll-a content showed a similar tendency. There was a highly significant relationship between the microcystin content of M. aeruginosa and the chlorophyll-a concentration in the culture during the incubation. Accordingly, the microcystin content of M. aeruginosa during incubation can be easily estimated and monitored by measuring the in vivo fluorescence changes in the culture.     

Effects of small scale fluid motion on bacterial growth and respiration

1. Laboratory experiments were conducted to investigate the effect of small‐scale turbulent motion on the growth and respiration of bacteria in an oscillating grid apparatus. The experiments were performed under a range of energy dissipation levels similar to those occurring in freshwater systems. 2. The results showed that small‐scale turbulent motion does have an effect on bacterial growth and respiration. A higher gradient in the dissolved oxygen time series, higher 5‐day biochemical oxygen demand values, increased bacterial abundance, increased bacterial specific respiration, higher bacterial growth rate and increased nutrient uptake were all observed when the energy dissipation rate in the water column was increased. 3. This has implications for traditional laboratory procedures that are used to characterise bacterial metabolic rates under stagnant fluid‐flow conditions, such as biochemical oxygen demand (BOD), which would be influenced by the effects of the small‐scale fluid motion inherent in aquatic environments. According to our results, BOD values in natural systems experiencing fluid motion would be higher than traditional bottle‐derived rates.     

Anaerobic growth and cyanide synthesis of Pseudomonas aeruginosa depend on anr, a regulatory gene homologous with fnr of Escherichia coli

Anaerobic growth of Pseudomonas aeruginosa on nitrate or arginine requires the anr gene, which codes for a positive control element (ANR) capable of functionally complementing an fnr mutation in Escherichia coli. The anr gene was sequenced; it showed 51% identity with the fnr gene at the amino acid sequence level. Four cysteine residues known to be essential in the FNR protein are conserved in ANR. The anr gene product (deduced Mr 27,129) was visualized by the maxicell method and migrated like a 32 kDa protein in gel electrophoresis under denaturing conditions. An anr mutant of P. aeruginosa constructed by gene replacement was defective in nitrate respiration, arginine deiminase activity, and hydrogen cyanide biosynthesis, underscoring the diverse metabolic functions of ANR during oxygen limitation. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, and Pseudomonas mendocina all had a functional analogue of ANR, indicating that similar anaerobic control mechanisms exist in these bacteria.     

Novel fermentation media for production of Bacillus thuringiensis subsp. israelensis

The production of Bacillus thuringiensis subsp. israelensis (deBarjac) (Bti) as a biopesticide is not cost-effective using existing fermentation technology. In this study, we explored the use of several less expensive alternative culture media (potato, common sugar, and Bengal gram) for the growth and production of Bti. Growth was obtained in all tested media and was comparable to that obtained in conventional medium (Luria-Bertani). Toxicity assays showed that the toxin produced from the novel growth media were effective in killing larvae of Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti and toxicity was comparable to that produced from Luria-Bertani medium. These observations suggest that potato can be used as a cheap source of culture medium for the production of Bti toxin in mosquito control programs.     

Oxygen regulation of nitrate uptake in denitrifying Pseudomonas aeruginosa.

Oxygen had an immediate and reversible inhibitory effect on nitrate respiration by denitrifying cultures of Pseudomonas aeruginosa. Inhibition of nitrate utilization by oxygen appeared to be at the level of nitrate uptake, since nitrate reduction to nitrite in cell extracts was not affected by oxygen. The degree of oxygen inhibition was dependent on the concentration of oxygen, and increasing nitrate concentrations could not overcome the inhibition. The inhibitory effect of oxygen was maximal at approximately 0.2% oxygen saturation. The inhibition appeared to be specific for nitrate uptake. Nitrite uptake was not affected by these low levels of aeration, and nitrite reduction was only partially inhibited in the presence of oxygen. The regulation of nitrate respiration at the level of transport by oxygen may represent a major mechanism by which the entire denitrification pathway is regulated in P. aeruginosa.     

Oxygen regulation of nitrate uptake in denitrifying Pseudomonas aeruginosa

Oxygen had an immediate and reversible inhibitory effect on nitrate respiration by denitrifying cultures of Pseudomonas aeruginosa. Inhibition of nitrate utilization by oxygen appeared to be at the level of nitrate uptake, since nitrate reduction to nitrite in cell extracts was not affected by oxygen. The degree of oxygen inhibition was dependent on the concentration of oxygen, and increasing nitrate concentrations could not overcome the inhibition. The inhibitory effect of oxygen was maximal at approximately 0.2% oxygen saturation. The inhibition appeared to be specific for nitrate uptake. Nitrite uptake was not affected by these low levels of aeration, and nitrite reduction was only partially inhibited in the presence of oxygen. The regulation of nitrate respiration at the level of transport by oxygen may represent a major mechanism by which the entire denitrification pathway is regulated in P. aeruginosa.     

Contribution of cell elongation to the biofilm formation of Pseudomonas aeruginosa during anaerobic respiration

Pseudomonas aeruginosa, a gram-negative bacterium of clinical importance, forms more robust biofilm during anaerobic respiration, a mode of growth presumed to occur in abnormally thickened mucus layer lining the cystic fibrosis (CF) patient airway. However, molecular basis behind this anaerobiosis-triggered robust biofilm formation is not clearly defined yet. Here, we identified a morphological change naturally accompanied by anaerobic respiration in P. aeruginosa and investigated its effect on the biofilm formation in vitro. A standard laboratory strain, PAO1 was highly elongated during anaerobic respiration compared with bacteria grown aerobically. Microscopic analysis demonstrated that cell elongation likely occurred as a consequence of defective cell division. Cell elongation was dependent on the presence of nitrite reductase (NIR) that reduces nitrite (NO(2) (-)) to nitric oxide (NO) and was repressed in PAO1 in the presence of carboxy-PTIO, a NO antagonist, demonstrating that cell elongation involves a process to respond to NO, a spontaneous byproduct of the anaerobic respiration. Importantly, the non-elongated NIR-deficient mutant failed to form biofilm, while a mutant of nitrate reductase (NAR) and wild type PAO1, both of which were highly elongated, formed robust biofilm. Taken together, our data reveal a role of previously undescribed cell biological event in P. aeruginosa biofilm formation and suggest NIR as a key player involved in such process.     

Mortality in adult tsetse, Glossina morsitans morsitans, caused by entomopathogenic bacteria

Mortality in adult tsetse, Glossina morsitans morsitans, caused by Pseudomonas aeruginosa, Serratia marcescens, Bacillus sphaericus, Bacillus cereus, Bacillus thuringiensis H-14, B. thuringiensis 1, B. thuringiensis 5, B. thuringiensis var. insraelensis, and Providentia rettgeri was determined. When bacteria were smeared on rabbit skin and tsetse allowed to feed only once on the contaminated area, mortality 8 days postingestion was significantly higher (P less than 0.01) in tsetse fed on P. aeruginosa, S. marcescens, B. thuringiensis 1, and P. rettgeri and increased when tsetse were allowed to feed for the second time on the contaminated skin. With this smear technique, however, mortalities were generally not remarkable. In artificial membrane feeding experiments using low concentrations of bacteria (-10(6)/ml of blood), the B. thuringiensis strains caused low mortality, except B. thuringiensis H-14, which caused 59% mortality. However, at this concentration, P. aeruginosa, S. marcescens, B. cereus, and P. rettgeri caused highly significant (P less than 0.01) mortality (64-96%). When higher concentrations of bacteria (10(7)/ml) were used, all the bacteria tested, except B. sphaericus, caused high mortality ranging from 70 to 98%. Thus, mortality depended on the species of bacteria, the dose ingested, and time postingestion.     

The effect of nitrogen limitation on catabolite repression of amidase, histidase and urocanase in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the synthesis of histidase, urocanase and amidase is severly repressed when succinate is added to a culture growing in pyruvate + ammonium salts medium. When growth is nitrogen-limited, catabolite repression by succinate of histidase and urocanase synthesis does not occur but succinate repression of amidase synthesis persists. Amidase synthesis is not regulated in the same way as histidase synthesis by the availability of other nitrogen compounds for growth. Growth of P. aeruginosa strain PACI in succinate + histidine media is nitrogen-limited since this strain is defective in a histidine transport system. When methyl-ammonium chloride is added to succinate + histidine media, growth inhibition occurs. Mutants isolated from succinate + histidine + methylammonium chloride plates were found to be resistant to catabolite repression by succinate even in ammonium salts media. It is suggested that the hut genes of P. aeruginosa may be regulated in the same way as in Klebsiella aerogenes, by induction by urocanate and activation by either the cyclic AMP-dependent activator protein or by glutamine synthetase.